Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR DETERMINING CELLULAR
RESPONSE TO STIMULI
Related Application
This patent document claims the benefit of priority of U.S. application serial
No. 60/639,152, filed December 22, 2004, and PCT application number
PCT/US2005/41946 filed on November 17, 2005, which applications are herein
incorporated by reference.
Background of the Invention
Taste transduction is one of the most sophisticated forms of
chemotransduction in animals. Gustatory signaling is found throughout the
animal
kingdom, from simple metazoans to the most complex vertebrates. Its main
purpose
is to provide a reliable signaling response to non-volatile ligands. Humans
typically
distinguish several perceptual taste qualities or modalities: sweet, sour,
salty, bitter
and umami. Each of these modalities is thought to be mediated by distinct
signaling
pathways mediated by receptors or channels, leading to receptor cell
depolarization,
generation of a receptor or action potential, and release of neurotransmitter
at
gustatory afferent neuron synapses.
Taste transduction in animals is mediated by specialized neuroepithelial
cells, referred to as taste receptor cells. These cells are organized into
groups of
about 40 to 100 cells to form taste buds. Taste buds contain precursor cells,
support
cells, and taste receptor cells. Receptor cells are innervated at their base
by afferent
nerve endings that transmit information to the taste centers of the cortex
through
synapses in the brain stem and thalamus. Taste buds are distributed into
different
papillae in the tongue epithelium. Circumvallate papillae, found at the very
back of
the tongue, contain hundreds to thousands of taste buds. By contrast, foliate
papillae, localized to the posterior lateral edge of the tongue, contain
dozens to
hundreds of taste buds. Further, fungiform papillae, located on the anterior
two-
thirds of the tongue, contain only a single or few taste buds, depending upon
the
species. Taste cells are also found in the palate and other tissues, such as
the
esophagus and the stomach.
Taste buds are ovoid structures and are primarily embedded within the
epithelium of the tongue. It is believed that taste transduction is initiated
at the
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apical portion of a taste bud at the taste pore, where microvilli of the taste
receptor
cells make contact with the outside environment. Various taste stimulants
cause
either depolarization (i.e., a reduction in membrane potential) or
hyperpolarization
(i.e., an increase in membrane potential) of taste cells and regulate
neurotransmitter
release from the cells at chemical synapses with afferent nerve fibers. The
primary
gustatory sensory fibers, which receive the chemical signals from the sensory
cells,
enter the base of each taste bud. Inter-cellular connections between taste
cells in the
same bud may also modulate the signals transmitted to the afferent nerve
fibers.
Molecules that elicit specific taste sensations are often referred to as
"tastants."
Although much is known about the psychophysics and physiology of taste cell
function, very little is known about the molecules and pathways that mediate
its
sensory signaling response.
In general, each taste modality is associated with particular types of
receptor
proteins expressed in some of the cells that form each taste bud. Genes
encoding
taste receptor proteins for sweet, bitter, umami and salty taste substances
have been
cloned from a variety of species, including humans. The nature of the coupling
of
stimulus-receptor interaction to a cellular response in the receptor cells has
also been
defined for some receptors. Some of these receptors have been used to develop
bioassays for use in identifying potential taste enhancers, blockers and
modifiers.
Although these "chip" based systems have the potential for high throughput
screening of large numbers of compounds, they do not incorporate the normal
cellular components of the taste signaling pathways that are required for
normal
receptor-response coupling. Consequently, these assays are best at providing
initial
information about binding of potential stimuli with a particular receptor.
They do
not, however, provide information about a subsequent cellular response, if
any, to
the test substance.
An alternative approach is to express cloned taste receptors in heterologous
cells, typically a mammalian cell line such as human embryonic kidney cells
(HEK293), and to measure changes in intracellular calcium induced by taste
stimuli.
This approach requires coupling between stimulus-receptor interaction and a
cellular
pathway leading to an increase in calcium, and it permits measurements in many
cells at once. The normal cellular organization of the taste receptor unit,
the taste
bud, however, is lost along with any processing of taste information occurring
between cells within the taste bud. This limitation is particularly important
in light
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of recent results suggesting that sweet and bitter receptors are localized in
taste cells
that do not directly communicate with afferent nerve fibers, but rather
communicate
with adjacent taste bud cells that are innervated.
Summar-Y of the Invention
Over the years substantial efforts have been directed to the development of
various agents that interact with taste receptors to mimic or block natural
taste
stimulants. Examples of agents that have been developed to mimic sweet tastes
are
saccharin, monellin, and the thaumatins. Many taste-mimicking or taste-
blocking
agents developed to-date are not suitable as food additives, however, because
they
are not economical, are high in calories, or are carcinogenic. Development of
new
agents that mimic or block the basic tastes has been limited by a lack of
knowledge
of the taste cell biology involved in the transduction of taste modalities.
Thus, there
is a continuing need for new products and methods involved in or affect taste
transduction.
The present invention provides a method for determining the functional
cellular response of a taste cell or taste cells contained in a section of
taste-bud
containing lingual epithelium (i.e., taste sensory cells in taste bud-
containing intact
epithelial tissue) to one or more stimuli. In the present invention, one
contacts tissue
such as taste tissue (such as taste bud-containing epithelial tissues or taste
papillae),
from an animal, with one or more stimuli, and quantitatively determines the
magnitude of at least one cellular signaling event initiated by the
stimulus/stimuli.
Multiple data values may be collected, such as at differing concentrations of
stimulus/stimuli and/or at different time points. The term "isolated taste bud-
containing intact epithelial tissue" refers to a tissue sample isolated from
an animal,
where the tissue has been removed such that the tissue, and the cells
contained in the
tissue, retains its integrity. For example, the isolated intact taste tissue
may be an
intact taste bud in an intact lingual epithelial tissue sample that includes
precursor
cells, support cells, and taste receptor cells such that the polarization of
the
epithelium is retained. Taste cells have an apical surface and a basal
surface. In
certain embodiments of the present invention, the stimuli contacts the apical
surface
of the taste cell or cells, but does not contact basal surface of the cell or
cells.
The invention provides methods of testing different taste stimuli, e.g.,
activators, inhibitors, stimulators, enhancers, agonists, and antagonists of
taste cells
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and tissues. As used herein, the term "taste cells" include neuroepithelial
cells that
are organized into groups to form taste buds of the tongue in structures known
as
papillae, e.g., foliate, fungiform, and circumvallate papillae (see, e.g.,
Roper et al.
(1989)). Taste cells also include cells of the palate and other tissues that
may
contain taste cells, such as the esophagus, the stomach, the gastrointestinal
tract or
other internal organs such as the liver or pancreas. The taste cells may be
taken
from a biological sample. Such samples include, but are not limited to, tissue
isolated from humans, mice, rats, and pigs. In addition to fresh tissue,
biological
samples may include sections of tissues such as frozen sections taken for
histological purposes. A biological sample is typically obtained from a
eukaryotic
organism, such as an insect, protozoa, bird, fish, reptile, or mammal (e.g., a
rat,
mouse,.cow, dog, pig, rabbit, chimpanzee, or human). Tissues include tongue
tissue
and isolated taste buds.
A "functional cellular response" in the context of the present invention
includes one or more cellular changes in response to any parameter that is
indirectly
or directly under the influence of the test stimulus, e.g., a functional,
physical or
chemical effect of the stimulus on the taste cell, cells, or tissue. A
functional
cellular response includes ligand binding, changes in ion flux, membrane
potential,
current flow, transcription, signal transduction, receptor-ligand
interactions,
messenger concentrations (including, but not limited to, cyclic AMP (cAMP),
inositol trisphosphate (IP3), or intracellular Ca++ or other positive and/or
negative
ions including, but not limited to, chloride, sodium, or protons), in vitro,
in vivo, and
ex vivo and also includes other physiologic effects, such as increases or
decreases of
neurotransmitter or hormone release.
As used herein, "determining a functional cellular response," means assaying
for an increase or decrease in a parameter in or on a cell that is indirectly
or directly
under the influence of the test stimulus, e.g., functional, physical and
chemical
effects. Such functional effects can be measured by any means known to those
skilled in the art, e.g., changes in optical or spectroscopic characteristics
(e.g.,
fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),
chromatographic, or solubility properties, patch clamping, voltage-sensitive
dyes,
whole cell currents, radioisotope efflux (or influx), inducible markers;
tissue culture
cell expression; transcriptional activation; ligand binding assays; voltage,
membrane
potential and conductance changes; ion flux assays; changes in intracellular
second
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messengers such as cAMP and inositol triphosphate (IP3); changes in
intracellular
calcium levels; neurotransmitter release, and the like. "Inhibitors,"
"activators," and
"modulators" are used to refer to inhibitory, activating, or modulating
molecules
identified using in vitro and in vivo assays for taste transduction, e.g.,
ligands,
agonists, antagonists, and their homologs and mimetics. Inhibitors are
compounds
that, e.g., bind to a cell or cell component (e.g., receptor), partially or
totally block
stimulation, decrease, prevent, delay activation, inactivate, desensitize, or
down
regulate taste transduction, e.g., antagonists. Activators are compounds that,
e.g.,
bind to a cell or cell component (e.g., receptor), stimulate, increase,
activate,
facilitate, enhance activation, sensitize or up regulate taste transduction,
e.g.,
agonists. Modulators include compounds that alter the interaction of a
polypeptide
with receptors or extracellular proteins that bind activators or inhibitor,
such as
kinases. Modulators include genetically modified, naturally occurring and
synthetic
ligands, antagonists, agonists, small chemical molecules and the like. Such
assays
for inhibitors and activators include, e.g., applying putative modulator
compounds,
in the presence or absence of tastants, and then determining the functional
effects on
taste transduction. Samples or assays comprising a cell in intact taste tissue
but that
are treated with a potential activator, inhibitor, or modulator are compared
to control
samples without the inhibitor, activator, or modulator to examine the extent
of
modulation. Positive control samples (e.g., a tastant without added
modulators) are
assigned a relative activity value of 100%. Samples treated with an inhibitor,
activator, or modulator are compared to control samples without the inhibitor,
activator, or modulator to examine the extent of inhibition, activation or
modulation.
Control samples (untreated with an inhibitor, activator, or modulator) are
assigned a
relative activity value of 100%. Inhibition is achieved when the activity
value
relative to the control is about 80%, optionally 50%, or 25, or even 0%.
Activation
is achieved when the activity value relative to the control is 110%,
optionally 150%,
optionally 200-500%, or 1000-3000%, or higher.
The present invention provides a method for simulating a taste, comprising
ascertaining the extent to which a cell in intact taste tissue interacts with
a tastant.
Interaction of a tastant with a cell in intact taste tissue can be determined
using any
of the assays described herein. The tastant can be combined with other
tastants to
form a mixture. If desired, one or more of the plurality of the compounds can
be
combined covalently.
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The present invention also provides a method wherein one or more control
tastants are tested against one or more test tastants, to ascertain the extent
to which a
sensory cell or group of sensory cells in taste bud-containing lingual
epithelial tissue
interacts with each control tastants, thereby generating a stimulation profile
for each
control tastants. These stimulation profiles may then be stored in a
relational
database on a data storage medium. The method may further comprise providing a
desired stimulation profile for a taste; comparing the desired stimulation
profile to
the relational database; and ascertaining one or more combinations of control
tastants that most closely match the desired stimulation profile. The method
may
further comprise combining control tastants in one or more of the ascertained
combinations to simulate the taste.
The invention also provides methods of screening for modulators, e.g.,
activators, inhibitors, stimulators, enhancers, agonists, and antagonists, of
tastants.
Such modulators of taste transduction are useful for pharmacological,
chemical, and
genetic modulation of taste signaling pathways. These methods of screening can
be
used to identify high affinity agonists and antagonists of taste cell
activity. These
modulatory compounds can then be used in the food and pharmaceutical
industries
to customize taste, e.g., to modulate the tastes of foods, beverages, or
drugs.
An "ingestible substance" is a food, beverage, or other comestible, or orally
administered products or compositions. A "flavor" herein refers to the
perception of
taste and/or smell in a subject, which include sweet, sour, salty, bitter,
umami, and
others. The subject may be a human or an animal. A "flavoring agent" herein
refers
to a compound or a biologically acceptable salt thereof that induces a flavor
or taste
in an animal or a human. A "flavor modifier" herein refers to a compound or
biologically acceptable salt thereof that modulates, including enhancing or
potentiating, and inducing, the tastes and/or smell of a natural or synthetic
flavoring
agent in an animal or a human. A "flavor enhancer" herein refers to a compound
or
biologically acceptable salt thereof that enhances the tastes or smell of a
natural or
synthetic flavoring agent.
"Savory flavor" herein refers to the savory "umami" taste typically induced
by MSG (mono sodium glutamate) in an animal or a human. "Savory flavoring
agent" or "savory compound" herein refers to a compound or biologically
acceptable
salt thereof that elicits a detectable savory flavor in a subject, e.g., MSG
(mono
sodium glutamate). A "savory flavor modifier" herein refers to a compound or
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biologically acceptable salt thereof that modulates, including enhancing or
potentiating, inducing, and blocking, the savory taste of a natural or
synthetic savory
flavoring agents, e.g., monosodium glutamate (MSG) in an animal or a human. A
"savory flavor enhancer" herein refers to a compound or biologically
acceptable salt
thereof that enhances or potentiates the savory taste of a natural or
synthetic savory
flavoring agents, e.g., monosodium glutamate (MSG) in an animal or a human.
A "savory flavoring agent amount" herein refers to an amount of a
compound that is sufficient to induce savory taste in a comestible or
medicinal
product or composition, or a precursor thereof. A fairly broad range of a
savory
flavoring agent amount can be from about 0.001 parts per million (ppm) to 100
ppm,
or a narrow range from about 0.1 ppm to about 10 ppm. Alternative ranges of
savory flavoring agent amounts can be from about 0.01 ppm to about 30 ppm,
from
about 0.05 ppm to about 15 ppm, from about 0.1 ppm to about 5 ppm, or from
about
0.1 ppm to about 3 ppm.
A "savory flavor modulating amount" herein refers to an amount of a
compound that is sufficient to alter (either increase or decrease) savory
taste in a
comestible or medicinal product or composition, or a precursor thereof,
sufficiently
to be perceived by a human subject. A fairly broad range of a savory flavor
modulating amount can be from about 0.00 1 ppm to 100 ppm, or a narrow range
from about 0.1 ppm to about 10 ppm. Alternative ranges of savory flavor
modulating amounts can be from about 0.01 ppm to about 30 ppm, from about 0.05
ppm to about 15 ppm, from about 0.1 ppm to about 5 ppm, or from about 0.1 ppm
to
about 3 ppm.
A "savory flavor enhancing amount" herein refers to an amount of a
compound that is sufficient to enhance the taste of a natural or synthetic
flavoring
agents, e.g., monosodium glutamate (MSG) in a comestible or medicinal product
or
composition. A fairly broad range of a savory flavor enhancing amount can be
from
about 0.001 ppm to 100 ppm, or a narrow range from about 0.1 ppm to about 10
ppm. Alternative ranges of savory flavor enhancing amounts can be from about
0.01
ppm to about 30 ppm, from about 0.05 ppm to about 15 ppm, from about 0.1 ppm
to
about 5 ppm, or from about 0.1 ppm to about 3 ppm.
Similar definitions are applicable to the other taste modalities of sweet,
sour,
salty, and bitter.
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The terms "isolated," "purified" or "biologically pure" refer to material that
is substantially or essentially free from components that normally accompany
it as
found in its native state. Purity and homogeneity are typically determined
using
analytical chemistry techniques such as polyacrylamide gel electrophoresis or
high
performance liquid chromatography.
A "label" or a "detectable moiety" is a material having a detectable physical
or chemical property, e.g., spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels include
fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the
like), radiolabels (e.g.,3H,'25I; 35S, 14C, or 32P) electron-dense reagents,
enzymes
(e.g., horse radish peroxidase, alkaline phosphatase and others), biotin,
digoxigenin,
or haptens and proteins that can be made detectable, e.g., by incorporating a
radiolabel into the peptide.
As used herein, the terms "a" and "an" can mean either single or plural.
Brief Description of the Fi2ures
This patent or application file contains at least one drawing executed in
color. Copies of this patent or patent application publication with color
drawing(s)
will be provided by the Office upon request and payment of the necessary fee.
Figure 1 is a schematic drawing of a taste bud. This figure was found on the
World Wide Web at cf.ac.uk/biosi/staff/jacob/teaching/sensory/papillae.gif.
Figure 2. Average fluorescence lifetime calculated from multiphoton
lifetime fluorescence imaging (MP-FLIM) system as function of calcium
concentration and dye concentration. Notice that the lifetime appears
invariant,
within experimental error, of dye concentration.
Figure 3. Average fluorescence intensity measured using the MP-FLIM
system as function of calcium concentration and dye concentration. Note that
the
emission intensity is a function of dye concentration and free calcium ion
concentration.
Figures 4A-4D. Multi-Photon Fluorescence Intensity (MP) and Lifetime
(MP-FLIM) images of a 1-micron diameter sphere taken with a 63x objective with
hardware zoom of 20 (-45 nm per pixel). The data were taken with the CLSM
scanning in the x/y plane and in the x/z plane; the relatively low resolution
in the x/z
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direction is consistent with literature values and optical theory. Figure 4A
is MP-
FLIM x/y; Figure 4B is MP-FLIM x/z; Figure 4C is MP x/y; and Figure 4D is MP
x/z.
Figures 5A and 5B. x/y plane Point Spread Function (PSF) rendered from
the MP x/y image in Figure 4 above. To match the conditions under which taste
cell
data are collected (in this case, 63x obj w. zoom 2), the original image had
to be
down-sampled by l Ox. The resulting PSF is on a 25x25 pixel grid (reduced from
the
original 256x256 grid). Figure 5A shows PSF at 63x, zoom2. Figure 5B shows PSF
at 63x, zoom20.
Figures 6A-6C. Preliminary deconvolution and denoising results. The Lucy-
Richardson algorithm was used to perform the deconvolution using a slightly
higher
resolution version (3x) of the PSF shown in Figure 5 above, along with noise
parameters calculated from the microsphere data shown in Figure 4 above to
reduce
noise amplification during the deconvolution process. This image was then
denoised (i.e, smoothing "points" while preserving "edges") using an intensity
gradient calculation method. Figure 6A shows a raw taste cell MP image. Figure
6B shows 100 liters of L-R algorithm with 3x finer-grid PSF. Figure 6C shows
DeNoising of L-R image using 4'h-order PDE method.
Figures 7A-7C. Example of time-series image stack registration. Note the
considerable drift to the top and left of the taste bud over the course of the
measurement (Unregistered #100) relative to its original position (Reference
#1).
The last image in the registered time-series stack (Registered #100) still
shows some
misalignment (note the position of the taste cell within the white ellipse in
the 3
images). Figure 7A shows unregistered #100, Figure 7B shows reference #1, and
Figure 7C shows Registered #100.
Figure 8. Mean intensity trends of the region enclosed by the white ellipse in
Figure 7. Note the dramatic loss of intensity in the unregistered stack: this
is due to
the taste cell drifting outside of the ROI over time. There is some loss of
intensity in
the registered stack, but much of this is due to photobleaching (the overall
intensity
of the field-of-view of this data drops by -10% over the course of the time-
series).
Figure 9. Time series multiphoton image of taste bud region during stimulus
with cycloheximide and sucralose. a) rest; b) cycloheximide addition; e)
refocus f)
sucralose stimulus. Notice that the sucralose stimulus results in significant
shortening of the calcium green lifetime around the taste cells. As the
calcium green
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lifetime associated with the epithelial cells remains essentially unchanged
during the
experiment, it is clear that he taste cells responded to the stimuli.
Figures l0A and l OB. Image field of view showing the two taste buds with
several cells in each loaded with dye (Figure l0A). The stimulus application
resulted in intracellular calcium increase in cell 1 and possibly cell 2
(Figure lOB).
However, the slope and baseline offset between cells make it difficult to
compare
responses. However, the alignment algorithm allowed individual cell ROI's to
be
defined for time series analysis.
Figures 11A-11C. Baseline corrected time series data for all seven cells in
the field of view during successive 20 mmol/L citric acid stimuli. The taste
buds
were exposed to artificial saliva (no stimulus) for 5 minutes between stimuli
to allow
for recovery. Note the strong response to the stimulus in cell 1 with lower
magnitude responses in other cells. The decrease in response to subsequent
stimulus
is most likely due to the lack of viability of the tissue or to adaptation.
Figure 11A
shows baseline corrected average intensity per cell, 20 mmol/L citric acid
stimulus
1; Figure 11B shows baseline corrected average intensity per cell, 20 mmol/L
citric
acid stimulus 2, and Figure 11C shows baseline corrected average intensity per
cell,
mmol/L citric acid stimulus 3.
20 Detailed Description of the Invention
Functional Organization of the Vertebrate Taste Bud
General Anatomic Features of the Peripheral Taste System
Taste buds contain 50-100 polarized neuroepithelial cells of several distinct
morphological and immunohistochemical types, at least some of which function
as
the receptor cells mediating taste signal transduction. (Witt and Reutter,
1996;
Finger and Simon, 2000). Three morphological classes of taste bud cells are
readily
distinguished in all vertebrates: dark (type I), light (type II) and basal or
progenitor
cells. However, intermediate cells with an appearance intermediate between
dark
and light cells (rodents), type III cells (initially in rabbits, but much less
obvious in
rodents), and Merkel-like basal cells (urodele amphibians) have also been
described
in some species. Additionally, classification of the cell types comprising the
taste
bud is further complicated by the presence of cells at different stages of
development
resulting from the replacement of taste cells throughout life.
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Type III cells were first described in rabbits (Murray, 1973). They resemble
type II cells in general morphological features, but have dense-cored vesicles
in the
cytoplasm around the nucleus (Takeda and Hoshino, 1975) and show serotonin-
like
immunoreactivity (LIR) (Uchida, 1985; Fujimoto et al., 1987; Kim and Roper,
1995). Initially, type IIl cells were not described in rats and mice, although
cells
with numerous large, dense-cored vesicles were described (Takeda and Hoshino,
1975). Also, a small subset of rodent taste cells have serotonin-LIR, which is
markedly enhanced by pre-treatment with the immediate serotonin precursor, 5-
HTP
(Kim and Roper, 1995; Bourne and Kinnamon, 1999). In rabbit, presynaptic taste
cells, i.e., those making afferent synapse onto nerve fibers, are type III
cells
(Murray, 1973; Royer and Kinnamon, 1991). Recent work, performed largely in
Kinnamon's lab, has begun classifying the intermediate cells of rodents, some
of
which are serotonergic, as type III cells. All three elongated taste cell
types in the
mouse, type I, intermediate, and type 11 cells, have been described to have
synaptic
contacts with nerve fibers (Kinnamon et al., 1985; Kinnamon et al., 1988;
Royer and
Kinnamon, 1994). These afferent synapses are of two structural types; macular
and
finger-like (Kinnamon et al., 1985, Kinnamon et al., 1988, Kinnamon et al.,
1993;
Royer and Kinnamon, 1994). While this is consistent with earlier work in fish
showing that both light and dark cells made synaptic contacts with nerve
fibers or
basal cells (Reutter, 1971). In rats, only intermediate or serotonergic (now
called
type III) cells make classical synaptic contacts with nerve fibers.
Immunohistochemistry of taste cells
The presence of serotonin-LIR in cells in rat taste buds that also have some
of the morphological features of intermediate cells has led to their tentative
classification as type III cells. In circumvallate taste buds, only taste
cells with
synapses (type III) show immunoreactivity for SNAP-25, a presynaptic membrane
protein (Yang et al., 2000). SNAP-25 is also present in most intragemmal and
perigemmal nerve fibers. This could mean that these fibers are "presynaptic",
i.e.,
efferent as well as afferent. For example, reciprocal, or two-way, synapses
have
been described in taste cells found in some amphibians. The presence of SNAP-
25
in taste bud nerve fibers could result from transport from the other end of
nerve
cells, which are presynaptic to higher order cells in the brain stem. In
contrast,
synaptobrevin, a vesicle-associated membrane protein (VAMP) believed to
function
with SNAP-25 to coordinate synaptic vesicle docking and release of
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neurotransmitter, appears to be present in both type III cells (also showing
NCAM-,
SNAP-25-, and serotonin-LIR) and a subset of type II cells (which also display
taste
signal transduction components such as PLC(32 and IP3R3 (Yang et al. 2004).
This
suggests that type I cells have "unconventional" synapses that have not been
recognized by TEM ultrastructure studies (see Clapp, et. al, 2004).
Taste transduction components associated with G-protein-coupled receptors,
IP3R3, PLC(32 , TRPM5 and Gy13, are expressed in a large subset of type II and
a
small subset of type III vallate taste cells in rat (Clapp et al., 2004). NCAM
is
present in many intermediate cells (type III cells) (Nelson and Finger, 1993).
5-HT-
LIR and PGP9.5-LIR are present in mutually exclusive subsets of type III
cells, but
PGP9.5 also in some type II cells (Yee et al., 2003). PLC signaling components
are
in a vast majority of type II cells and small subset of type III cells. Brain
derived
neurotrophic factor (BDNF) appears to be in all type III cells, but only a
small
subset of type II cells (Yee et al. 2003). Only type III cells in rat have
conventional
synapses with nerve fibers.
Functional Responses of the Taste System
Although a variety of functional cell markers have been used in rodent taste
buds, the results are somewhat confusing with regard to classical
morphological cell
types. A variety of subsets of presumably functionally distinct taste cells
appears to
exist. Using antigen H (blood type antigen) to mark type I cells, antigen A
for type
II cells and NCAM for type III cells, Medler, et al. (2003) identified the
types of
voltage-gated currents in CV and foliate taste cells of mouse. Classified in
this way,
all type I cells and many type II cells displayed small voltage-gated sodium
and
potassium currents and no calcium currents. A subset of type II cells and all
type III
cells had large Na and K currents as well as voltage-gated Ca currents.
Unexpectedly, the subset of type II cells that were gustducin positive lacked
Ca
currents. These results are consistent with the idea that type III cells have
synapses
with nerve fibers, but that type II cells with G-protein-coupled receptors do
not have
conventional chemical synapses.
Also, Lyall et al., 2005 and Lyall et al., 2004 have implicated a novel
vanilloid receptor variant in mediating the amiloride-insensitive component of
the
salt taste response in mammals. However, these data do not completely rule out
a
role for epithelial sodium channels (ENaCs) in salt taste responses.
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There is growing evidence from physiological studies (Finger et al., 2005;
Baryshnikov et al. 2003) that ATP plays a role as a neurotransmitter in the
taste
system. Knockout mice for P2X and/or P3X ionotropic receptors have greatly
compromised taste responses. Baryshnikov et al., in contrast, show that P2Y
(metabotropic) ATP receptors are present on mouse taste cells and are
activated by
ATP via a PLC, IP3 cascade that releases internal Ca, followed by an influx of
Ca.
P2X receptors are present on nerve fibers and could mediate responses to ATP
released from taste cells. P2Y receptors on taste cells, however, could
mediate
neuromodulatory responses to ATP from other taste cells or to ATP released
from
efferent nerve endings.
There is also the recent work with neuropeptides Y (NPY ) cholecystokinin
and other neuropeptides (Herness et al., 2005) These substances are released
by
taste cells ad appear to have both autocrine effects on the releasing cell and
endocrine effects on neighboring cells. They presumably act as neuromodulators
in
setting the responsiveness or sensitivities of taste receptor cells. The
overlap of
NPY with cholecystokinin or vasoactive intestinal peptide was 100%. Given the
opposite effects of NPY and the other peptides on cellular responsiveness,
this
suggests a push-pull system in a given cell.
The present invention provides a method for determining the functional
cellular response of taste bud-containing lingual epithelial tissue to one or
more
stimuli. The tissue to be tested includes live, intact taste cells. The tissue
may be
obtained from an animal in the form of a biopsy, or may be obtained
immediately
after sacrifice of an animal (e.g., pig). The tissue is prepared for further
study. In
one embodiment, the harvested tissue is prepared into slices. In another
embodiment, individual papillae are isolated. In another embodiment,
individual
taste papillae with functional taste buds are isolated. The isolated tissue is
then
placed either in or on a solid substrate, such as a microscope cover slip. The
solid
substrate may be coated with a tissue-adhering coating. The tissue may then be
placed in a tissue chamber that allows for perfusion of oxygenated cell media
and
stimuli.
The tissue to be tested may be contacted with a label or a detectable moiety.
For example, the tissue may be contacted with a fluorescent dye. In certain
embodiments, a dye may be loaded into a tissue or cell or group of cells.
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The tissue to be tested is contacted with one or more test taste stimuli, such
as activators, inhibitors, stimulators, enhancers, agonists, and antagonists
of taste
cells and tissues such that exposure of the taste cells to stimuli is isolated
to the
apical ends of the taste cells through the taste pore. The contacting may be
achieved
by point delivery by proximate delivery of stimulus via micropipette to the
taste pore
region, or by bulk delivery via perfusion of a known stimulant concentration
(i.e.,
bathing the tissue in stimulant). The stimulus may be in contact with the
tissue
briefly (e.g., for a few seconds), or for an extended period of time (e.g.,
for a few
minutes). Further, the stimulus may be in contact with the tissue for a period
of
time, discontinued for a period of time, and then reapplied. This cycling can
be
repeated numerous times. Alternatively, the stimulus may be applied at one
concentration, and then at a later time period, the stimulus may be applied at
another
concentration, either higher or lower.
One then detects and/or quantitatively determines the magnitude of at least
one cellular signaling event initiated by the stimulus/stimuli. A wide variety
of
techniques and technologies may be used. Examples include wide field
fluorescence
imaging using native fluorescence or fluorophores with specific binding
properties,
confocal laser scanning microscopy (e.g., multiphoton confocal laser scanning
microscopy). Multiple data values may be collected, such as at differing
concentrations of stimulus/stimuli and/or at different time points.
In the method of the present invention, one quantitatively determines the
cellular signaling in response to the one or more stimuli. For example, one
could
determine the cellular signaling response in the absence of a stimulus,
determine the
response in the presence of the stimulus, and even then determine the response
after
the stimulus has been removed. In other words, different temporal measurements
can be determined and different concentration measurements can be determined.
Live, intact taste cells/buds/tissue
The approach of the present invention permits direct documentation of lateral
interactions among cells within taste buds. Several approaches are presented
that
allow for response measurement of taste tissue system. The taste bud tissue
may be
extracted from live test subjects from the species under study or from
sacrificed
animals. Taste cells may be obtained from appropriately sectioned taste
papillae and
used as thick sections (200 micrometers thick) that would contain a majority
of the
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taste cell and bud structure in the section. The slice preparation provides
experimental access to nearly intact taste buds, which retain most of the
morphological and functional organization of in situ taste buds.
A taste bud slice preparation also provides the ability to determine if taste
responses are modified by lateral and/or sequential processes in cells within
the taste
buds. For example, dyes sensitive to membrane potential can be used to measure
concurrent electrical responses in taste cells. Membrane dyes can also be used
to
measure synaptic vesicle recycling to determine which cells release
neurotransmitter
in response to a taste stimulus. Dyes such as Lucifer yellow can be used to
assess
the effects of electrical coupling between taste cells on signal processing.
For example, in the case of porcine tissue, an intact tongue is procured from
a local slaughterhouse and immediately placed in chilled storage (0 C) for
transport
to the laboratory. The tongue is then examined and sections of tissue are
removed
containing fungiform papillae from the lateral portion of the tongue. The
tissue
sections in this case contain not only epithelial tissue, but taste cells and
the
connective tissue underneath the papillae. The section, or sections, removed
in this
manner are placed in an appropriate oxygenated storage solution, as described
in the
references such as Danilova et al., 1999. In another embodiment, a biopsy is
removed from a live animal (e.g., pig) and immediately prepared for
examination.
In one embodiment, the tissue section is prepared (e.g., using a vibrating
microtome) into a slice section of thickness between 100 and 200 micrometers.
This
is accomplished using chilled tissue and storage solution such that the
sections are
cut with minimal cutting artifacts. The individual tissue sections are then
placed
onto a microscope cover slip coated with a tissue-adhering coating or protein
(collagen is typically used). This cover slip is then attached, via suitable
removable
adhesive or highly viscous, non-reacting grease, to a tissue chamber that
allows for
perfusion of oxygenated cell media and stimuli. This presentation is then
ready to
be loaded with appropriate cell signaling dye and imaged for response to
stimulus as
a slice section. Alternately, the slice section may be prepared, in a manner
similar to
that above, after the papillae tissue is loaded with an appropriate signaling
dye.
An alternate approach is to utilize intact preparations of taste tissue
excised
from the tongue as papilla tissue, with epithelial cells and an intact taste
pore located
on the top of the tissue. For example, a subject animal (e.g., pig or human)
is
anaesthetized and biopsied along the lateral portion of the tongue, resulting
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removal of a block of tissue one to two millimeters on a side. This tissue is
then
placed directly in chilled storage solution and prepared, under a dissecting
microscope, into a tissue sample containing a single fungiform papilla. This
is then
transferred to a cover slip such that the apical face of the taste bud is
pointing up
from the cover slip, exposing the taste pore and surrounding epithelial
tissue, and the
basolateral portion of the papilla is attached to the coverslip over a small
hole,
allowing for perfusion of the tissue from the basolateral side. The tissue may
be
adhered to the coverslip via a coating of collagen or the application of
tissue cement
to the edges of the papilla in contact with the coverslip. This sample is thus
prepared for subsequent loading of appropriate signaling dye. Examples of
general
taste tissue preparation and loading protocols and results are given in the
following
references: Lindemann, 1996; and Caicedo et al., 2000.
Taste tissue (taste buds or taste papillae) can be isolated from a wide
variety
of animals. For instance, rodents are subjects of studies involving
stimulation of
taste sensory systems in the work presented in Caicedo et al., 2000. In
addition,
feline specimens have been used for taste response studies, according to the
following references: Boudreau et al., 1973; Boudreau et al. 1977; Boudreau et
al.
1971; Krimm et al. 1998; and Boudreau et al. 1985. Additionally, canines are
subjects of studies involving stimulus of taste sensory systems in the work
presented
in the following references: Kumazawa et al., 1990; Kumazawa et al., 1991;
Kumazawa et al., 1990; Nakamura et al. 1990; and Nakamura et al. 1991.
The isolation of taste sensory systems (taste buds or taste papillae) from
taste
tissue is not limited to mammalian subjects. For instance, species of fish
have been
the subject of taste system study, according to the following representative
references: Caprio, J. 1975; Brand et al., 1991; Zviman et al. 1996; Hayashi
et al.,
1996; Finger et al., 1996; and Ashworth 2004.
Imaging system consisting offluorescence imaging of cellular signaling events
The imaging of cellular signaling events is performed using a variety of
techniques and technologies. The prevailing tool in this area of endeavor is
optical
microscopy of fluorescent structures and features in cells, which encompasses
a
broad spectrum of specific equipment and approaches. First, wide field
fluorescence
imaging using both native fluorescence and addition of fluorophores with
specific
binding properties to cell species is a widely used tool. In addition,
confocal laser
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scanning microscopy (CLSM) of fluorescently labeled cells with ultraviolet or
visible light excitation is a tool gaining increased use in cell studies.
Alternatively,
confocal laser scanning microscopy of fluorescently labeled cells with
multiphoton
excitation using a near-infrared laser is a methodology that allows for deeper
tissue
sampling and reduced tissue damage due to the optical effects exploited in
multiphoton excitation of fluorescent molecules.
Fluorescence is the result of a three-stage process that occurs in certain
molecules (generally polyaromatic hydrocarbons or heterocycles) called
fluorophores or fluorescent dyes. A fluorescent probe is a fluorophore
designed to
localize within a specific region of a biological specimen or to respond to a
specific
stimulus.
In stage one, a photon of energy hvEx is supplied by an external source such
as an incandescent lamp or a laser and absorbed by the fluorophore, creating
an
excited electronic singlet state (S,'). This process distinguishes
fluorescence from
chemiluminescence, in which the excited state is populated by a chemical
reaction.
In stage two, the excited state exists for a finite time (typically 1-10
nanoseconds). During this time, the fluorophore undergoes conformational
changes
and is also subject to a multitude of possible interactions with its molecular
environment. These processes have two important consequences. First, the
energy
of Sl' is partially dissipated, yielding a relaxed singlet excited state (Si)
from which
fluorescence emission originates. Second, not all the molecules initially
excited by
absorption (stage one) return to the ground state (So) by fluorescence
emission.
Other processes such as collisional quenching, fluorescence resonance energy
transfer (FRET) and intersystem crossing (see below) may also depopulate S.
The
fluorescence quantum yield, which is the ratio of the number of fluorescence
photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a
measure
of the relative extent to which these processes occur.
During stage three of the fluorescence excitation/emission process, a photon
of energy hvEM is emitted, returning the fluorophore to its ground state So.
Due to
energy dissipation during the excited-state lifetime, the energy of this
photon is
lower, and therefore of longer wavelength, than the excitation photon hvEx.
The
difference in energy or wavelength represented by (hvEx - hvEM) is called the
Stokes
shift. The Stokes shift is fundamental to the sensitivity of fluorescence
techniques
because it allows emission photons to be detected against a low background,
isolated
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from excitation photons. In contrast, absorption spectrophotometry requires
measurement of transmitted light relative to high incident light levels at the
same
wavelength.
The entire fluorescence process is cyclical. Unless the fluorophore is
irreversibly destroyed in the excited state (an important phenomenon known as
photobleaching, see below), the same fluorophore can be repeatedly excited and
detected. The fact that a single fluorophore can generate many thousands of
detectable photons is fundamental to the high sensitivity of fluorescence
detection
techniques. For polyatomic molecules in solution, the discrete electronic
transitions
represented by hvEx and hvEM are replaced by rather broad energy spectra
called the
fluorescence excitation spectrum and fluorescence emission spectrum,
respectively.
The bandwidths of these spectra are parameters of particular importance for
applications in which two or more different fluorophores are simultaneously
detected (see below). With few exceptions, the fluorescence excitation
spectrum of
a single fluorophore species in dilute solution is identical to its absorption
spectrum.
Under the same conditions, the fluorescence emission spectrum is independent
of
the excitation wavelength, due to the partial dissipation of excitation energy
during
the excited-state lifetime. The emission intensity is proportional to the
amplitude of
the fluorescence excitation spectrum at the excitation wavelength. Additional
information regarding optical processes involved in fluorescence can be found
in
Lakowizc, J.R., 1999.
Fluorescence microscopy is very useful due to the inherent specificity
afforded by the application of fluorescent labels to specific cell targets. As
an
example, Molecular Probes Corporation markets and sells a complete library of
standardized fluorescent tags functionalized to allow for specific assays and
tagging
experiments. For instance, calcium sensitive dyes respond to changes in
intracellular calcium ion concentration by changing the excitation or emission
properties of the dye. For the calcium ion indicators fura-2 and indo-1 the
free and
ion-bound forms of fluorescent ion indicators have different emission or
excitation
spectra. With this type of indicator, the ratio of the optical signals can be
used to
monitor the association equilibrium and to calculate ion concentrations.
Ratiometric
measurements eliminate distortions of data caused by photobleaching and
variations
in probe loading and retention, as well as by instrumental factors such as
illumination stability. Lifetime measurements of fluorophores in different
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environments allow for quantitative measurement of the environment
perturbation,
regardless of differences in dye distribution and optical scattering, making
this an
attractive mode of measurement in living tissue.
Wide-field fluorescence microscopy is very widely used to obtain both
topographical and dynamic information. It relies on the simultaneous
illumination
of the whole sample. The source of light is usually a mercury lamp, giving out
pure
white light. Optical filters are then used in order to select the wavelength
of
excitation light (the excitation filter). Excitation light is directed to the
sample via a
dichroic mirror (i.e., a mirror that reflects some wavelengths but is
transparent to
others) and fluorescent light detected by a camera (usually a CCD camera).
Thus
both the illumination and detection of light covering the whole visual field
of the
chosen microscope objective is achieved simultaneously.
Alternatively, laser scanning confocal microscopy (LSCM or CLSM,
abbreviated CLSM in this document) may be used to excite and collect
fluorescence
from a system under study. Measuring fluorescence by CLSM differs from wide-
field fluorescence microscopy in a number of ways. First, the light source is
one or
more laser(s). This has two consequences. First, the excitation light
bandwidth is
determined by the source, not the excitation filter and thus is much narrower
than in
wide field fluorescence microscopy (2-3 nm rather than 20 - 30 nm). Second, in
order to illuminate the whole visual field, the laser beam has to be rapidly
scanned
across the area in a series of lines, much like a TV image is generated. The
fluorescence detected at each point is measured in a photomultiplier tube
(PMT),
and an image is built up. This method of illumination has enormous advantages
in
that it is possible to illuminate selected regions of the visual field
allowing complex
photobleaching protocols to be carried out to investigate the rates of lateral
travel of
fluorophores and for the excitation of different fluorophores in different
regions of
the same cell. The major difference between fluorescence microscopy and CLSM
is
the presence of a pinhole in the optical path. This is a device that removes
unwanted, out-of-focus fluorescence, giving an optical slice of a 3-
dimensional
image. In one embodiment of the imaging protocol, excitation light incident on
the
sample excites the object of interest, and gives high-resolution fluorescent
image
with a minimum amount of haze or out-of-focus light reaching the detector. In
order
to obtain such an image, the pinhole is placed in front of the detector
photomultiplier
tube and blocks the passage of this out-of-focus light into the PMT. This
means that
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the only light to enter the PMT, and thus be detected, comes from near the
focal
plane of the objective lens of the microscope. As this is taken across the
area of the
sample, it produces an image that is a slice through the object and
surrounding
material. This is known as "optical slicing" and allows the observer to see
inside the
object of interest. This gives clear images, with fine detail observable.
In addition to the above advantages, by altering the focus of the microscope,
images can be obtained at different depths. Each image is called a z-section,
and can
be used to reconstruct an image of the 3-dimensional object. As an analogy,
this
technique is like cutting an object into slices, and then stacking the slices
back on
top of each other to reconstruct the shape of the object. This principle can
similarly
be achieved using multiple z-sections. If images are "stacked" on top of one
another
in the correct order, a single three-dimensional image of the object can be
generated.
In an alternative embodiment, multiphoton CLSM may be used, where
multiphoton excitation of the fluorophores of interest is achieved. As stated
earlier,
the energy of emitted fluorescent light is less than that of the incident
light. Also,
fluorophores that absorb red light do not emit green fluorescence. This holds
true
for almost all fluorescent applications. Under appropriate conditions,
however, the
generation of high energy fluorescence using low energy incident light is
achieved
by delivering multiple photons of excitation light to the same point in space
in a
sufficiently short time that the energy effectively is summed and so acts as a
higher
energy single photon. The arrival of the first photon causes the electron to
become
excited, but not sufficiently to reach a more stable state. This excess energy
is lost
very quickly, but if a second photon is delivered rapidly enough, the electron
acts as
if a high-energy single photon has been delivered, resulting in fluorescence
emission
from the focal point fluorophores.
The timescale of electronic excitation is incredibly short. The second photon
must arrive within <0.1 femtoseconds, so photons must be delivered in rapid
succession. The power required to deliver such a rapid continuous stream of
photons into a particular position in space, however, is enormous, up to about
1-
Terawatt/cmz. In order to prevent damage to biological samples, pulsed lasers
are
typically used. A typical multiphoton excitation laser is the
Titanium:Sapphire laser,
which delivers pulses of photons of about 100 femtoseconds duration separated
by
about 12-40 nanoseconds, at wavelengths ranging from -700 to 1000 nanometers.
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I nus, although the pulses oY light are of extremely high intensity, the
average power
delivered to the sample is relatively low.
Several advantages exist with regard to employing multiphoton excitation in
fluorescence imaging. First, light from the high intensity red to near
infrared laser
scatters less efficiently than lower intensity blue light, so objects of
interest can be
imaged in thicker sections of tissue than in conventional CLSM. Thicker tissue
slices are likely to be healthier, and the cells being observed are less
likely to have
been damaged in the preparation of the sample. Second, the lower overall
energy of
the excitation light means that less phototoxic damage is caused during
viewing and
less photobleaching is seen, extending the time that cells can be observed.
Third,
multiphoton CLSM is innately confocal, i.e., no pinhole is required.
Excitation of
the fluorophore can occur only where the two photons can interact. Given the
quadratic nature of the probability of two photons interacting with the
fluorophore in
the necessary timescale, excitation occurs only in the focal plane of the
objective
lens. This provides cleaner images. Finally, the high repetition rate pulsed
laser
used in multiphoton CLSM is uniquely suited to performing rapid fluorescent
lifetime measurements. Thus, a cell with an appropriate calcium sensitive dye,
like
calcium green dextran, is imaged using multiphoton fluorescence lifetime
imaging
with a properly configured CLSM to yield deep tissue measurement of
intracellular
calcium release in a receptor cell, such as a taste receptor cell.
Additional information regarding biological applications of fluorescence
microscopy in cell signaling studies may be found in the following references:
Gratton et al. 2003; Koester et al., 1999; Konig, K. et al. 1997; Liu et al.,
2003.
Quantitative stimulus delivery system applied to live tissue
The ability to provide known concentrations of taste stimuli to the
preparation under precise temporal and spatial control is critical to
quantitative
determination of cellular signaling in response to stimulus. The delivery of
stimuli
may be by any one or several methods. In one embodiment of the present
invention,
for example, the delivery of stimuli is achieved by point delivery by
proximate
delivery of stimulus via micropipette to the taste pore region. In another
exemplary
embodiment, the delivery of stimuli is achieved by bulk delivery via perfusion
of
known stimulant concentration, effectively bathing the tissue in stimulant.
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In one embodiment of a proximate delivery approach, slices of taste papillae
containing taste buds are mounted on glass cover slips coated with adhesive
protein.
The cover slips form the bottom of the recording/perfusion chamber, which is
attached to the stage of the microscope. A continuous background flow of
oxygenated, physiological saline (e.g., Tyrode solution) is supplied to the
chamber
from a temperature-controlled reservoir via an independently heated perfusion
line.
The direction of flow across the slice is oriented from the base to the apical
region of
the taste bud. Taste stimuli or mixtures of stimuli are applied from an
independent,
8-line, temperature-controlled perfusion pencil. Each of seven lines are
attached to
pressurized solution reservoirs via miniature (normally-closed) solenoid
valves. The
8cn line is attached via a normally-open solenoid valve to a regulated vacuum
line
and vacuum trap. The solenoids are under computer control so that a stimulus
valve
opens while the vacuum line is simultaneously closed for a predetermined time.
This device is positioned with the delivery tip close to and slightly distal
to the
apical end of the taste bud. This allows small puffs of up to seven different
stimuli
to be applied just to the apical, receptive ends of the taste cells. By
adjusting the
pressure in the stimulus reservoirs, the position of the stimulus delivery
tube, and the
rate of background perfusion, stimuli can be confined to just the apical
surfaces of
the taste bud cells. It is desirable to prevent activation of cellular
pathways in the
basolateral membranes of the taste bud cells that normally do not interact
with taste
stimuli on the surface of the tongue, which could lead to erroneous
conclusions
about taste response properties. The vacuum line in the stimulus delivery
manifold
is typically closed slightly after activation of the chosen stimulus valve and
acts to
prevent mixing of stimuli by evacuating solution leaking from closed lines and
for
priming the selected delivery tube. Solutions are continuously removed from
the
chamber by a vacuum line set to provide a constant fluid level and to pull
solution
without "jitter" or "slurping" at the solution surface.
In an alternative embodiment, pieces of tongue epithelium containing taste
buds are removed from the underlying tissue using gentle enzymatic treatment
or via
surgical removal. The epithelium is mounted in a trans-epithelial chamber that
allows independent perfusion of the basolateral (serosal) and apical (mucosal)
surfaces. This permits the normal polarity of the epithelium and the taste
buds in it
to be maintained. The basolateral chamber is perfused with oxygenated Tyrode
solution that has been pre-heated to minimize out-gassing and formation of
bubbles.
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Manipulations of basolateral ion channels and transports are manipulated by
application of Tyrode solution containing pharmacological agents by switching
to
other perfusate reservoirs through a bank of valves. The apical chamber is
used
either in an open configuration with a dipping objective or closed, with a
cover glass
attached to the top of the chamber. In either case, stimuli is applied from
temperature-controlled reservoirs attached to the apical chamber via computer-
controlled solenoid valves attached to a manifold attached to the chamber with
a
short length of tubing. This system is either gravity fed via constant flow
syringes
or through pressurized reservoirs. In either case, up to 24 independent
stimuli can
be applied to the apical chamber and then rinsed out under computer control
with the
present system. In an alternative stimulus delivery arrangement, the stimulus
is
delivered via a sample injection loop placed inline with the apical perfusion
line.
The volume of the injection loop is controlled via loop length, and under
static flow
rate conditions, the stimulus may be reproducibly introduced into the sample
chamber at a known time and using a known concentration.
Collection of stimulus/response data structure from imaging/stimulus system
A stimulus/response data structure may be collected from a live cell
preparation in the following general manner. A cell and/or tissue sample is
obtained
and mounted in a suitable oxygenated bath to allow for live cell imaging. The
cell/tissue is exposed to signaling-sensitive fluorescent dye such that the
intracellular
matrix (or membrane, in the case of membrane potential-sensitive dyes, and
recycling synaptic vesicles, in the case of synaptic activity markers) is
loaded with
dye. The cell and/or tissue sample is transferred to the imaging platform that
contains the stimulus system and a perfusion chamber, and supporting
apparatus, to
allow for constant perfusion of the sample during the experimental data
collection.
Image data is collected from the cell and/or tissue sample, using appropriate
image
modality and acquisition parameters to provide a baseline or standard resting
response image data matrix prior to quantitative stimulus.
Stimulus or series of stimuli are applied in the form of one or more solutions
of known concentrations of stimulus prepared in surrogate or synthetic saliva
matrices. The application of stimulus is proximate to the taste pore and is
administered via a micropipette system having control of stimulus flow and
pipette
position. Alternatively, the application of stimulus is administered via the
perfusion
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medium, thus bathing the cell and/or tissue sample in a known concentration of
stimulus. Before, during, and after the application of known stimulus to the
cell
and/or tissue sample, image data are collected in such a manner that a number,
if not
all, of the cells comprising the taste bud are represented in the field of
view captured
by the imaging system. The image data are collected in such a manner that data
corresponding to multiple time points for each phase (before, during, and
after
stimulus) are collected. Image data collected at each time point may be
comprised
of two or three spatial dimensions.
By way of example, an intact pig tongue is procured from a local
slaughterhouse and immediately placed in chilled storage (4 C) for transport
to the
laboratory. The tongue is then examined and sections of tissue are removed
containing fungiform papillae from the lateral portion of the tongue. The
tissue
sections in this case contain not only epithelial tissue, but also taste cells
and the
connective tissue underneath the papilla. The section, or sections, removed in
this
manner are placed in an appropriate oxygenated Tyrode storage solution.
The tissue section is prepared, using a vibrating microtome, into a slice
section of thickness between 100 and 200 micrometers. This is performed using
chilled tissue and storage solution such that the sections are cut with
minimal cutting
artifacts. The individual tissue sections are then placed onto a microscope
cover slip
coated with a tissue-adhering coating or protein (collagen is typically used).
This
cover slip is then attached, via suitable removable adhesive or highly
viscous, non-
reacting grease, to a tissue chamber that allows for perfusion of oxygenated
cell
media and stimuli. This presentation is then iontophoretically loaded with a
dye,
such as 5 mg/mL calcium green dextran (3000 MW), and imaged for response to
glutamate stimulus as a slice section. The loading of calcium sensitive dye is
verified via standard wide field epifluorescence microscopy. The tissue slice
preparations are then transferred to the measurement chamber on the recording
microscope, such as a multiphoton confocal laser scanning microscope with
epifluorescence optical filters designed to allow for multiphoton excitation
of the
dye and recording of emission above 530 nm. A continuous background flow of
oxygenated, physiological saline (Tyrode solution) is supplied to the chamber
from a
temperature-controlled reservoir via an independently heated perfusion line.
The
direction of flow across the slice is oriented from the base to the apical
region of the
taste bud. Taste stimuli or mixtures of stimuli are applied from an
independent, 8-
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line, temperature-controlled perfusion pencil. Each of seven lines is attached
to
pressurized solution reservoirs via miniature (normally-closed) solenoid
valves. The
8'n line is attached via a normally open solenoid valve to a regulated vacuum
line
and vacuum trap. The solenoids are under computer control so that a stimulus
valve
opens while the vacuum line is simultaneously closed for a predetermined time.
This device is positioned with the delivery tip close to and slightly distal
to the
apical end of the taste bud.
For stimulus one, at time = zero, the perfusion and stimulus chambers allow
flow with no stimulus, and one or more image frames are acquired whereby the
intensity of the generated fluorescence is acquired on a per pixel basis along
one
plane through the tissue, capturing several, if not all, of the cells in the
tissue in the
field of view. These frames are referred to as the baseline frames, prior to
stimulus.
After 2-5 minutes of perfusion with no stimulus, a low concentration stimulus
of
glutamate (0.05 mmol/L) in artificial saliva perfusion medium is applied via a
single
pipette channel proximate to the taste pore for a predefined length of time,
such as
120 seconds. An initial response to the stimulus is recorded via the
fluorescence
imaging system upon activation of the stimulus channel. Subsequently, two or
more
additional image frames are recorded during the stimulus time period,
generating a
series of frames collected during stimulus. The stimulus is then discontinued
and
the perfusion continues with no stimulus present, effectively rinsing stimulus
from
the region of interest around the taste pore. After the stimulus is removed,
and at
time intervals corresponding to 60 seconds, additional frames are recorded
using the
fluorescence imaging system in order to develop three frames of image data
corresponding to the resting, or post stimulus, response of the taste system
under
study after stimulus one.
For stimulus two, the perfusion and stimulus chambers allow flow with no
stimulus, and single image frame is acquired whereby the intensity of the
generated
fluorescence is acquired on a per pixel basis along one plane through the
tissue,
capturing several, if not all, of the cells in the tissue in the field of
view. This frame
is referred to as the baseline frame, prior to stimulus for stimulus two and
may be
different in intensity from the baseline stimulus one frame. After 2-5 minutes
of
perfusion with no stimulus, a low concentration stimulus of glutamate (0.25
mmol/L) in artificial saliva perfusion medium is applied via a single pipette
channel
proximate to the taste pore for a predefined length of time, such as 120
seconds. An
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initial response to the stimulus is recorded via the fluorescence imaging
system upon
activation of the stimulus channel. Subsequently, two additional image frames
are
recorded during the stimulus time period, generating a series of frames
collected
during stimulus. The stimulus is then discontinued and the perfusion continues
with
no stimulus present, effectively rinsing stimulus from the region of interest
around
the taste pore. After the stimulus is removed, and at time intervals
corresponding to
60 seconds, additional frames are recorded using the fluorescence imaging
system in
order to develop three frames of image data corresponding to the resting, or
post
stimulus, response of the taste system under study after stimulus two.
For stimulus three, the perfusion and stimulus chambers allow flow with no
stimulus, and single image frame is acquired whereby the intensity of the
generated
fluorescence is acquired on a per pixel basis along one plane through the
tissue,
capturing several, if not all, of the cells in the tissue in the field of
view. This frame
is referred to as the baseline frame, prior to stimulus for stimulus three and
may be
different in intensity from the baseline stimulus two frame. After 2-5 minutes
of
perfusion with no stimulus, a low concentration stimulus of glutamate (0.75
mmol/L) in artificial saliva perfusion medium is applied via a single pipette
channel
proximate to the taste pore for a predefined length of time, in this case 120
seconds.
An initial response to the stimulus is recorded via the fluorescence imaging
system
upon activation of the stimulus channel. Subsequently, two additional image
frames
are recorded during the stimulus time period, generating a series of frames
collected
during stimulus. The stimulus is then discontinued and the perfusion continues
with
no stimulus present, effectively rinsing stimulus from the region of interest
around
the taste pore. After the stimulus is removed, and at time intervals
corresponding to
60 seconds, additional frames are recorded using the fluorescence imaging
system in
order to develop three frames of image data corresponding to the resting, or
post
stimulus, response of the taste system under study after stimulus three. The
collection of these three time-dependent stimulus/response image data sets
results in
single image data structure that is then used to generate a quantitative
relationship
between stimulus and taste tissue response as determined by intensity-based
calcium
green dextran fluorescence measurements of changes in intracellular calcium.
An alternative embodiment of this experiment is as follows. A subject pig is
anaesthetized and biopsied along the lateral portion of the tongue, resulting
in
removal of a block of tissue five millimeters on a side. This tissue is then
placed
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directly in chilled storage solution and prepared, under a dissecting
microscope, into
a tissue sample containing a single fungiform papilla. This is then
transferred to a
collagen-coated cover slip such that the apical face of the taste bud is
pointing up
from the cover slip, exposing the taste pore and surrounding epithelial
tissue. This
cover slip is then attached, via suitable removable adhesive or highly
viscous, non-
reacting grease, to a tissue chamber that allows for perfusion of oxygenated
cell
media and stimuli. This presentation is then iontophoretically loaded with 5
mg/mL
calcium green dextran (3000 MW) and imaged for response to glucose stimulus as
a
slice section. The loading of calcium sensitive dye is verified via standard
wide
field epifluorescence microscopy by application of dye to the apical end of
the taste
bud through the taste pore.
The intact tissue preparations are then transferred to the measurement
chamber on the recording microscope, in this case a multiphoton confocal laser
scanning microscope with epifluorescence optical filters designed to allow for
multiphoton excitation of the dye and recording of time correlated picosecond
fluorescence lifetime emissions above 530 nm in such a manner as to allow for
fluorescence lifetime images to be generated. This is accomplished via a time
correlated single photon counting detector attached to the output port of the
confocal
laser-scanning microscope (such as a Leica MP-FLIM system).
The epithelium is mounted in a trans-epithelial chamber that allows
independent perfusion of the basolateral (serosal) and apical (mucosal)
surfaces.
This permits the normal polarity of the epithelium and the taste buds in it to
be
maintained. The basolateral chamber is perfused with oxygenated Tyrode
solution
that has been pre-heated to minimize out-gassing and formation of bubbles.
Manipulations of basolateral ion channels and transports can be manipulated by
application of Tyrode solution containing pharmacological agents by switching
to
other perfusate reservoirs through a bank of valves. Stimuli are applied from
temperature-controlled reservoirs attached to the apical chamber via computer-
controlled solenoid valves attached to a manifold attached to the chamber with
a
short length of tubing.
For stimulus one, at time = zero, the perfusion and stimulus chambers allow
flow with no stimulus, and single image frame is acquired whereby the
intensity of
the generated fluorescence is acquired on a per pixel basis along one plane
through
the tissue, capturing several if not all of the cells in the tissue in the
field of view.
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This frame is referred to as the baseline frame, prior to stimulus. After 5-7
minutes
of perfusion with no stimulus, a low concentration stimulus of glutamate (0.05
mmol/L) in artificial saliva perfusion medium is applied via a single pipette
channel
proximate to the taste pore for a predefined length of time, in this case 240
seconds.
An initial response to the stimulus is recorded via the fluorescence imaging
system
upon activation of the stimulus channel. Subsequently, two additional image
frames are recorded during the stimulus time period, generating a series of
frames
collected during stimulus. The stimulus is then discontinued and the perfusion
continues with no stimulus present, effectively rinsing stimulus from the
region of
interest around the taste pore. After the stimulus is removed, and at time
intervals
corresponding to 120 seconds, additional frames are recorded using the
fluorescence
imaging system in order to develop three frames of image data corresponding to
the
resting, or post stimulus, response of the taste system under study after
stimulus one.
For stimulus two, the perfusion and stimulus chambers allow flow with no
stimulus, and single image frame is acquired whereby the intensity of the
generated
fluorescence is acquired on a per pixel basis along one plane through the
tissue,
capturing several if not all of the cells in the tissue in the field of view.
This frame
is referred to as the baseline frame, prior to stimulus for stimulus two and
may be
different in intensity from the baseline stimulus one frame. After 5-7 minutes
of
perfusion with no stimulus, a low concentration stimulus of glutamate (0.25
mmol/L) in artificial saliva perfusion medium is applied via a single pipette
channel
proximate to the taste pore for a predefined length of time, such as 240
seconds. An
initial response to the stimulus is recorded via the fluorescence imaging
system upon
activation of the stimulus channel. Subsequently, two additional image frames
are
recorded during the stimulus time period, generating a series of frames
collected
during stimulus. The stimulus is then discontinued and the perfusion continues
with
no stimulus present, effectively rinsing stimulus from the region of interest
around
the taste pore. After the stimulus is removed, and at time intervals
corresponding to
120 seconds, additional frames are recorded using the fluorescence imaging
system
in order to develop three frames of image data corresponding to the resting,
or post
stimulus, response of the taste system under study after stimulus two.
For stimulus three, the perfusion and stimulus chambers allow flow with no
stimulus, and single image frame is acquired whereby the intensity of the
generated
fluorescence is acquired on a per pixel basis along one plane through the
tissue,
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capturing several if not all of the cells in the tissue in the field of view.
This frame
is referred to as the baseline frame, prior to stimulus for stimulus three and
may be
different in intensity from the baseline stimulus two frame. After 5-7 minutes
of
perfusion with no stimulus, a low concentration stimulus of glutamate (0.75
mmol/L) in artificial saliva perfusion medium is applied via a single pipette
channel
proximate to the taste pore for a predefined length of time, in this case 240
seconds.
An initial response to the stimulus is recorded via the fluorescence imaging
system
upon activation of the stimulus channel. Subsequently, two additional image
frames
are recorded during the stimulus time period, generating a series of frames
collected
during stimulus. The stimulus is then discontinued and the perfusion continues
with
no stimulus present, effectively rinsing stimulus from the region of interest
around
the taste pore. After the stimulus is removed, and at time intervals
corresponding to
120 seconds, additional frames are recorded using the fluorescence imaging
system
in order to develop three frames of image data corresponding to the resting,
or post
stimulus, response of the taste system under study after stimulus three. The
collection of these three time-dependent stimulus/response image data sets
results in
single image data structure that is then used to generate quantitative
relationship
between stimulus and taste tissue response as determined by emission lifetime-
based
calcium green dextran fluorescence measurements of changes in intracellular
calcium.
Quantitative relationship development between image data and stimulus
Once the stimulus-dependent image data are collected, a quantitative
relationship between the stimulus applied and the response of the cellular
system is
developed. This may be achieved by the following stepwise general approach.
First, the image data is standardized with respect to feature position (x,y
axis),
feature intensity (z axis), and feature time (t axis). This includes, e.g.,
defining onset
of stimulus application (initial time or Ti) and the disappearance of stimulus
response (final time or Tf) for each series of images associated with a
particular
stimulus application; registering each image within such a series such that
spatial
information in each image in the series may be correlated to subsequent images
(Ti+1 to Tf) within that series (and collection of series for a sequence of
stimulus
applications); and normalizing the baseline and dynamic ranges for the
responses
between each image within such a series.
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Next, a Region or Regions of Interest (ROIs) are defined within each of the
standardized images that contain phenomena related to the magnitude of the
applied
stimulus. The information within such ROIs is used to extract vector or scalar
quantities that represent the independent variables in the comparison step.
Independent variables from the standardized data ROIs are thein extracted.
For magnitude or concentration measurements, this includes reducing the
collection
of pixel values for a given image ROI to a scalar quantity. For temporal
measurements, this includes reducing time-dependent intensity changes in
lifetime
or intensity ratio within a series of image ROIs to vector quantities. For
spatial
measurements, this includes reducing, e.g., diffusion events across ROIs in
the (x,y)
space to vector quantities. This may include multiple scalar/vector
extractions for
more than one ROI within each image. After extraction, the independent
variables
are then grouped into a single vector to represent a particular stimulus
application.
Next, the known stimulus applications (dependent variables) are compared
against their corresponding independent variable vectors. The comparison may
include elements of classification, regression, or qualitative procedures that
are used
to rank the data according to the applied stimuli. Such procedures define a
model
which, when applied to an independent variable vector collected on a
stimulated
sample in the future, yield a classification estimate or predicted stimulus
magnitude.
In addition, this procedure yields estimates of statistical quantities such as
correlation coefficients for the independent vector components and confidence
limits
for the classification estimate or stimulus magnitude prediction.
By way of example, image data standardization is begun by applying an
intensity correction to each image within the set so that each image's
response to a
pre-defined internal standard condition is the same. For example, an inert
fluorescent dye of known concentration is injected into the field of view
along with
the applied stimulus, and the response of this dye is defined to have a
certain
magnitude. Alternatively, the image data set is normalized to maximum and
minimum values of intensity and this normalization is applied to subsequent
images
for comparison.
Following this step, the set of images are corrected for random movement of
objects within the field of view during data acquisition. The mathematical
procedure of correcting arbitrary differences between a set of
multidimensional
collections of values is called Procrustes Rotation. Such procedures determine
a
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linear transformation (translation, reflection, orthogonal rotation, and
scaling) of the
points in a matrix Y (or in this case a digital image) to best conform them to
the
points in another matrix X. Different methods, such as promax, orthomax, or
varimax rotations can be applied, which differ mainly in the criterion they
use to
estimate differences in the alignment fit. Standardizing the images with
respect to
time includes defining the onset of stimulus application (initial time or Ti)
and the
disappearance of stimulus response (final time or Tf) for every collected set
of
images. Ti could be defined as the first image in the series to show any data
point
response above a pre-defined baseline or un-stimulated condition, and Tf could
be
defined as the first image after Ti to register no data point responses above
the
baseline condition (i.e., the imaged field of view has returned to an "un-
stimulated"
state).
Having corrected the raw image data, the informative ROIs of the images is
then isolated. This can be done manually by having a trained operator input
image
coordinates or interactively draw perimeters around ROIs in a reference image.
A
more rigorous way is to calculate the magnitude of response for each data
point in
the set of images between Ti and Tf. Those values with maximum responses below
a certain threshold are discarded as "noise" or "background." Further, the
remaining
data points are grouped into similar types according to the pattern of their
response
to the applied stimuli as a function of time. For example, certain regions of
the field
of view might record transitory "spikes" in response along the time axis,
while
others demonstrate a steady-state increase in response throughout the entire
period
from Ti to Tf. This pattern recognition can also be done manually.
Alternatively,
quick mathematical routines such as Principal Components Analysis (PCA) or
Cosine Correlation Analysis (CCA) can be used to define the different types of
informative data points in the image. Likewise, if temporal information is not
important for quantification, the average or summed response of each data
point
could be calculated across the time axis of a given set of images. Groups of
interconnected data points with similar response levels are then defined as
individual
ROIs. Regardless of the procedure used, the ROI definitions need to be done
only
once, since all of the images in a set (and across sets collected for the same
field of
view) have been aligned with respect to each other, so that an ROI in any
given
image is in the exact same place on all other images within a set, and among
different image sets.
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Depending upon how the ROIs were defined, the information within them
may have to be transformed into scalar or vector quantities to make it
appropriate for
comparison to the magnitude of the applied stimulus. For temporal
measurements,
this includes reducing time-dependent intensity changes in lifetime or
intensity ratio
within a series of image ROIs to vector quantities. The most straightforward
way to
do this is to average together all of the time profiles within a given ROI to
form one
vector of time responses. For ROIs that include spatial gradients, such as a
cell
membrane that demonstrates diffusion of a stimulus from its outer to inner
boundaries over the time-span of the stimulus application, diffusion gradients
could
be calculated mathematically by measuring the differences between nearest-
neighborhood data points from image to image over the time-span. This is
similar to
calculating a derivative via finite-differences. The diffusion pathway is
identified as
the chain of data points that exhibits the largest negative residual
differences among
subsequent image subtractions. For example, one subtracts the cell membrane
ROI
of image Ti+1 from image Ti. The data point with the largest negative
magnitude in
this residual image (which should be on the outer boundary of the membrane)
will
be the start of the diffusion gradient. Then one subtracts image Ti+2 from
image
Ti+1, and finds the data point with the largest negative residual that is
close in space
to the previous maximum residual points. This step is repeated for all of the
remaining images up to Tf (or until the maximum negative residual "wave" has
traversed the entire width of the membrane). Then, one calculates the rate of
residual intensity transfer in units of distance/time across the width of the
membrane. This procedure thereby results in a scalar quantity (diffusion rate)
that is
compared to, for example, stimulus magnitude or stimulus type. Furthermore,
several such waves from different parts of the membrane can be calculated in
order
to determine an average diffusion rate. Note that several other quantities can
be
calculated for a set of images from different ROIs that exhibited different
types of
information (i.e., magnitude/static, spatial, and/or temporal). After
calculating these
values, they are then grouped into a single set of numbers (a vector) to
represent the
independent variables that are to be compared to a particular stimulus
application.
The comparison may include elements of classification, regression, or
qualitative procedures that are used to rank the data according to the applied
stimuli.
Such procedures define a model which, when applied to an independent variable
vector collected on a sample stimulated in a similar manner in the future,
yields a
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classification estimate or predicted stimulus magnitude. In addition, this
procedure
yields estimates of statistical quantities such as correlation coefficients
for the
independent vector components and confidence limits of, for example, a
stimulus
type classification estimate or stimulus magnitude estimate. An example of a
classification type of comparison is to perform PCA on a collection of vectors
calculated from several image sets that were exposed to different types of
stimuli
(e.g., sweet, bitter, salt). The PCA scores of this reduced data set clusters
in the
principal components space according to stimulus type. As an example of a
quantitative regression application, the level of applied sweet stimulus in an
unknown sample could be predicted using a multivariate correlation algorithm
such
as non-iterative Partial Least Squares (PLS). PLS is applied to a set of
response
vectors calculated from several applications of known levels of sweetness in
order to
construct a pattern of correlations between the calculated response vectors
and their
corresponding level of sweet stimulus. This pattern of correlations
constitutes a
mathematical model that can be applied to future response vectors in order to
yield a
prediction of applied sweetness.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light
thereof
will be suggested to persons skilled in the art and are to be included within
the spirit
and purview of this application and scope of the appended claims.
Example 1 -- Multiphoton Fluorescence Lifetime Imaging System
Characterization: Response to Calcium Green Dextran Solutions of Known Free
Calcium Concentration
The present inventors made use of a multiphoton lifetime fluorescence
imaging microscopic (MP-FLIM) system capable imaging calcium dynamics within
taste cells in intact, live porcine taste tissue. The approach made use of a
calcium
sensitive fluorescent dye (calcium green-1 dextran MW=3000), where the dye
emission properties (fluorescence lifetime and intensity) were perturbed by
intracellular calcium concentration changes in the nanomolar to millimolar
range.
Multiphoton-excited fluorescence, in this case, made use of a pulsed near-
infrared
laser to excite fluorescence from a visible fluorophore via a two or three
photon
process. This non-linear optical process allowed for deep tissue imaging of
fluorophore emission with minimal cell damage from the near-infrared laser due
to
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lack of absorption outside of the focal plane and absence of cytotoxic
compounds
that are generally formed as a result of photo degradation of dye molecules
during
standard ultraviolet calcium imaging.
The approach taken in this work made use of the changes in intracellular
calcium concentration to indicate and quantitate the response of the gustatory
system
(taste bud) to stimuli. A key attribute of the MP-FLIM approach was the
ability of
the system to provide quantitative calcium concentration information from a
calcium
sensitive dye and its response to changes in free calcium ion in-vivo. In
order to
determine a quantitative relationship between stimulus and response, the
response of
the system to changes in bulk free calcium ion needed to be determined.
The inventors determined the relationship between the fluorescence lifetimes
of a two-photon excited calcium green dextran dye and varying concentrations
of
free calcium in buffer. In addition, the effect of dye concentration in buffer
was
studied in order to determine if variations in cell loading would cause
uncertainty in
lifetime determination. Calcium green-I dextran was chosen as the calcium
sensitive dye for this work for the following reasons: (1) Calcium green-1 is
a
visible-light excitable calcium indicator (Ex. 505/Em. 532) with a KD for
calcium in
the appropriate range (-540 nM) for taste cell studies; (2) displays about a
100 fold
increase in fluorescence intensity upon calcium ion binding; (3) has been used
for
measurements of relative changes in calcium (intensity) in taste cells in
response to
stimuli in rodents; and (4) a dextran-conjugated dye greatly reduces problems
with
sequestration of the dye in subcellular compartments and dye binding to
cellular
proteins. It also reduces transport of the dye out of the cell, which is a
common
problem in taste cells loaded with AM (membrane permeant) dyes.
Calcium buffers (CaEGTA) and calcium green-1 dextran (CaGD) were
purchased from Molecular Probes. The calcium buffers provide free Ca+2 over a
nanomolar to millimolar concentration range in a matrix closely matching the
pH
and ionic strength of the cytosol. The lifetimes and relative contributions of
these
bound and unbound decays are determined by fitting multi-exponential curves to
the
instrumental data. Buffer kit #2 (0-39 micromoles/L free Ca+z) was used in
this work
to determine the response of calcium green dextran to low calcium
concentrations.
Initial work was done with Buffer kit #3, but the range of free Ca+2 (0-1000
micromoles/L) was too high for the dye chosen. Calcium ion concentration is
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approximateiy U. i micromoies/L in resting cell's cytoplasm and in the
increases to
the micromolar range upon stimulus (Pollard, 2002).
Samples were analyzed in a random order during each experiment using the
MP-FLIM system and a quartz solution cell. Initial experiments were performed
with 5-micromoles/L dye in the calcium buffer solutions, and these data are
presented here first. Each sample produced two full fields of view (256 x 256)
where each image pixel contains a decay curve. As the calcium green dextran
was
present in bound and unbound forms, double exponential fits were performed
using
SPCImage (version 2.7.7238.0). The double exponential decays were calculated
for
each sample on the entire field of view, generating 65,536 values for each
field of
view. The average decay for each field of view was calculated as a sum of the
per
pixel lifetime values weighted by the amplitudes (contributions) of each
lifetime to
each pixel. Table 1 details the results for the calculation of the unbound dye
fluorescence lifetime. The average il from the measurements is 405.6 ps, with
a
sample standard variance of approximately 1.0 ps.
Table 1. Ca+z results, calculation of short (unbound) lifetime component of
the
decay for calcium green dextran MW =3000, 5 micromoles/L dye.
Standard
Sample Mean lifetime, Mean unbound deviation, il,
i ps lifetime, il, ps
ps
0 a 1 617.8 403.3 144.1
0 a 2 603.6 405.5 109.3
0_b_1 623.0 406.9 95.7
0 b 2 619.1 404.4 90.85
The low standard deviation demonstrates the repeatability of the bulk
measurement system for sample-to-sample averaged over the entire field of
view.
Individual samples exhibit an average il variation of 109 ps per field of
view,
meaning the pixel to pixel variation under these fitting conditions is on the
order of
109 ps. The balance of the calcium buffer data were analyzed using the
SPCImage
software under similar conditions as given in Table 1, with the exception that
the
short lifetime was fixed to 405.6 ps in the model parameters settings box. In
addition, the zero Ca+z buffer average lifetimes were recalculated using the
fixed
short lifetime value. Due to instabilities in the software interface, the
short lifetime
was designated as i2 in the fit parameters box. The average lifetime
calculation
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results for the series of buffers are given in Table 2. A second order
polynomial fit
of average lifetime in picoseconds versus calcium concentration in
micromoles/L for
a calcium concentration range from 0 to 0.602 micromoles/L yields the
relationship:
ta,,g=-3047 c2+ 4472 c + 601 , R2=0.9976; RMSE = 30.9 ps.
Table 2. Average lifetimes calculated for each buffer, 4 fields of view per
buffer,
short lifetime fixed to 405.6 ps. The sample standard deviation is the
standard
deviation calculated from the average lifetimes from four fields of view. The
pixel
variability is a measure of variation within in a field of view. Dye
concentration was
5 micromoles/L.
mean Std, Std, pixel Mean
Free Ca, lifetime, sample variation, photon
micromoles/L ps variation, ps counts
ps
0.000 587.8 18.1 152.19 2042
0.017 653.0 4.6 103.77 2524.9
0.038 761.3 5.3 74.947 2846.8
0.065 880.9 7.1 52.66 3414.7
0.100 1068.6 8.7 58.68 3702.1
0.150 1215.6 28.4 44.02 4551.2
0.225 1463.2 25.2 48.03 5354.9
0.351 1750.4 15.2 59.69 6355
0.602 2200.8 25.8 51.77 7968.8
1.350 2841.9 4.9 46.31 10294
39.000 3081.9 3.0 82.27 13113
A significant reported advantage of fluorescence lifetime measurements is
that the dependence of fluorescence lifetime on calcium concentration is
insensitive
to dye concentration provided the dye itself is at low enough concentrations
so as to
not buffer the calcium ion in the cell or the cuvette. In order to test this
property in
the calcium green dextran system, additional sample sets were made up with 2.5-
micromoles/L dye and 9.9-micromoles/L dye in the buffer solutions. The
variations
in lifetime data and intensity data with dye concentrations are given in
Figure 2 and
Figure 3, respectively. The lifetime data appears to be invariant with dye
concentration, where the average standard deviation (variation in lifetime
with dye
concentration at each calcium concentration) for the lifetime data on the
order or 25
ps. In contrast, the intensity variability is significant, where the intensity
of the
fluorescence is dependent upon the calcium concentration and the dye
concentration.
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Finally, calcium green-1 dissociation constants were calculated for the
various dye concentrations using lifetime and intensity-based data sets. The
Kd, or
dissociation constant, of a chelator (indicator) is the binding constant for
the
complexation of the dye and calcium ion and is expressed in units of moles/L.
The =
lifetime data exhibited a Kd standard deviation (n=3) of 27.3 nmol/L, and the
intensity data exhibited a Kd standard deviation (n=3) of 71.4 nmol/L (see
Table 3).
Table 3. Variation in dissociation constant with dye concentrations, Kd
calculated
based on intensity or lifetime data. The 2.5 and 9.9 micromole/L data were
collected
on March 11, 2005, and the 5.0-micromole/L dye data were collected on March 7,
2005. The intensity-based dissociation constant calculations from March 11,
2005
exhibit greater variation than the values calculated from lifetime data.
Dye concentration Kd, nmol/L, Kd, nmol/L, Lifetime
Intensity Data Data
2.5 micromoles/L 482.5 342.0
5.0 micromoles/L 520.1 329.1
9.9 micromoles/L 382.0 289.5
The following observations and conclusions are made based on the
calibration and characterization experiments detailed in this section. The MP-
FLIM
system provides reproducible fluorescence lifetime measurements of lifetimes
ranging from <400 ps to >3000 ps, standard deviation of 25-30 ps. Calcium
green
dextran indicator fluorescence intensity and lifetime increase reproducibly
with
calcium concentration. The dissociation constant Kd for CaGD was calculated
under various conditions using lifetime and intensity data and found to be
consistent
with that reported by the supplier (Molecular Probes, Inc.). The Kd ranged
from 320
to 520 nM, with the lifetime determination providing a more reproducible Kd
than
that for the intensity-based method. For low micromolar indicator
concentrations,
fluorescence lifetime is independent of indicator concentration, while the
fluorescence intensity depends significantly on indicator concentration.
Example 2 -- Quantitative Algorithm Development and Estimation of System
Optical Performance
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There are a number of tasks involved to ensure precise and robust
measurement of changes that occur in the image data obtained from the MP-FLIM
system. First, an attempt should be made to mitigate artifacts induced by the
optical
path and detector on the "true" image data. In a spatially invariant system
(i.e., all
the pixels in the field of view experience the same optical and detector
effects), such
artifacts can be classified as either resolution loss (blurring) or recorded
pixel
intensity errors (noise). Second, because the image data are recorded in a
flow cell,
the image features may move over time, the process of image registration
compensates for such movement by re-aligning the images in a given time-series
with respect to a reference state (usually the first image in the series).
Proper image
registration is necessary when using region-of-interest (ROI) methods to
quantify
intensity changes in image features over time. Finally, an additional
consideration
in FLIM data is the fast and accurate calculation of lifetimes from the
measured
decays in each image pixel. Limitations in the currently used software
included with
the FLIM system will require incorporation of algorithms into MATLAB to
perform
these tasks alongside the artifact reduction routines mentioned above.
Estimation of System Optical Performance
An optical system is characterized by its Point Spread Function (PSF), which
measures the effect on a point source of travel through the optical path and
measurement by the optical detector. A PSF can be inferred from optical theory
(usually assumed to be a symmetric Gaussian function), but a more accurate
characterization can be done using fluorescent microspheres of known diameter.
Collecting micrographs of isolated spheres under identical optical and
environmental
conditions as the sample to be studied results in a picture of how these
variables
affect a standard object. The size of the microsphere is known, and if it is
small
enough relative to the optical and digital resolution of the imaging system,
it can be
considered a homogeneous point source. Therefore, an image of this object can
be
treated as the actual PSF of the system (see Figure 4).
The only further requirements of a PSF for use in calculations are that it
preserves scale upon inversion (i.e., its elements sum to one), and that it is
spatially
invariant (i.e., it is plotted on a symmetric grid, with its center of mass in
the exact
center of the grid). In addition, background noise was subtracted from the
microsphere image (so that "empty" pixels had a value of zero) and the central
intensity feature was smoothed slightly to eliminate "roughness" due to
detector
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noise. Because the original PSF image was taken at the highest resolution the
highest resolution that the optical system was capable of (63x objective with
hardware zoom of 20), this processed image was then down-sampled to match the
conditions under which a particular sample image was collected prior to
deconvolution. Deconvolution is the process by which the function of the
optical
system, as represented by the PSF, is removed from the sample image (see
Figure 5).
Quantitative Algorithm Development
The Lucy-Richardson (L-R) algorithm was chosen as the means by which to
deconvolve the PSF from the sample images. This algorithm is relatively fast
and
robust, and is considered "state-of-the-art" in the imaging community. The L-R
routine employs certain "damping" parameters to help mitigate this; these
parameters include the mean and variance of background noise (calculated from
the
microsphere images used to formulate the PSF) under assumptions of Poisson
statistics (usually valid in "counting" experiments like digital imaging).
However,
so far it seems necessary to employ some of form of denoising (smoothing)
along
with the deconvolution in order to get the best possible feature resolution
and noise
elimination. A technique involving relative smoothing based upon intensity
gradients works well at preserving "real" deconvolved features from amplified
noise. As with the L-R algorithm, this method operates relatively quickly in
MATLAB. This combination of deconvolution and denoising produces images that
seems to make feature boundaries more distinct while reducing "speckle" (see
Figure 6).
Aside from the instrumental artifacts, there are considerable movement
artifacts in the image data. This problem of image registration is well known
and
can be effectively solved for many types of scale, perspective, and field of
view
differences between two images of the same scene. However, this robust
approach
requires a user to define reference points in each image that are used to
construct
spatial transformation matrix that is used to project the candidate image onto
the axis
space of the reference image. Since taste cell time-series usually consist of
at least
100 images, this degree of user intervention is impossible. Therefore, image
registration in this case must rely on "blind" iterative trials of coordinate
shifts of
each image in the stack relative to the first image. This process is actually
fairly
quick for stacks in which the maximum movement of a given image is less than
10
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pixels in either direction, requiring perhaps 2 minutes to register a stack of
100
images (see Figure 7) using a P4 2.8 GHz computer with 4 GB RAM.
Currently the registration process has some errors for images requiring large
corrections (more than 10 pixels on an axis), but these are less than 5
pixels, and
make ROI operations on individual features feasible (see Figure 8). The errors
are
probably due to how the criterion for success is defined as well as real
changes in
feature intensity over a time-series stack.
While all of the above work applies to MP-FLIM as well as MP data, MP-
FLIM data also requires determining the lifetimes of the fluorescent entities
in an
image as well as the relative intensity contributions of those lifetimes to
each pixel
in an image. So, for a 2-component system measured in a 256 x 256 image with
50
time points per pixel, there will be 256 x 256 x 3 = 1.97 x 105 parameters
that need
to be estimated over 3.7x106 data points. Without making any assumptions, this
task
requires several hours, and the results may be very inconsistent for low S/N
data (as
is often the case with MP-FLIM, especially in dynamic systems). Speed and/or
accuracy of the solution can be greatly increased by using a priori lifetime
values
that are kept constant during the fitting, so that only the relative intensity
contributions need to be fit (the so-called "lifetime invariant" approach). If
these a
priori lifetimes are accurate, and the instrument response function is
accurately
known, then incorporating these assumptions into an iterative fitting routine
will
give good, fast results. Even then, however, dramatic increases in speed can
be
found by segmenting the image into features based on binned intensity or very
quick
lifetime fits. This results in usually less than 10 features, which are then
fit to yield
initial lifetime and relative intensity estimates. Using these initial
estimates, each
pixel is then fit independently. These fits usually require far less
iterations to
converge, since the initial feature-based estimates are likely to be close to
the final
fitted value for all the pixels within a given region. In fact, this approach
has been
found to be comparable in speed and accuracy with the lifetime-invariant
approach
on high S/N images. In addition, it has been found to be equally accurate with
only
a moderate decrease in speed for low S/N images where the time invariant
approach
often converges to unrealistic parameter estimates (Pelet, 2004). Finally,
since this
feature segmentation requires user interaction at the start of the algorithm,
the
inventors of this approach show that an image division initialization produces
fits
that are somewhat slower and slightly less accurate, but that still yields
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results for various image types while having the additional quality of being
fully
automated.
Calibration and Prediction Considerations
To establish a functional correspondence between type and concentration of
stimulus as a dependent variable and taste bud response as an independent
variable,
the information in the MP & MP-FLIM images must be reduced into a set of
variables that make up the input into a prediction equation to solve for
either a
stimulus type-response or a modifier perturbation response. Mathematically,
the
question is this: what will the various components of the regression equation
f(x)=y
be? In a standard regression approach to what would be expected from initial
stimulus-response experiments, y is a matrix consisting of between one and
five
quantities related to tastant concentrations in the stimulus solutions, x is a
matrix of
the measured features in the MP images, and "f' is the regression relationship
between x and y. Ideally, f would be a linear combination of the x's, which
would
make the problem solvable by the widest variety of linear and linearizable
techniques, e.g., PLS variants.
Example 3 -- Development of Tissue Preparation Protocols
The utilization of porcine taste tissue in taste research is a new approach in
taste research at the cellular level. Several researchers have performed
behavioral
studies with pigs (Kare, et. al, 1965) and one has performed
electrophysiological
recordings from nerve fibers and whole nerves innervating the peripheral taste
system of pigs exposed to various stimuli (Danilova, 1999). These works
provide a
basis for the similarity between human and porcine taste responses, in bulk,
but
provide no knowledge regarding the anatomy and physiology of the porcine taste
system. Therefore, the inventors embarked on a multi-faceted approach to
develop
knowledge of the porcine peripheral taste system in support of the project
system
development and the overall project goals of discovering taste modifiers.
In support of this work, microscopic assays were performed to ascertain the
anatomy and physiology of the porcine taste bud system. These assays include:
1)
Scanning electron microscopy for high-resolution surface anatomy of the taste
structure; 2) Transmission electron microscopy for ultrastructural studies,
including
wide field microscopy through the sample preparation phase to assess tissue
quality;
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3) Immunocytochemical studies on fixed and prepared tissue section for
determining
the presence of specific classes of cellular machinery related to
intracellular
signaling and synaptic function; and 4) Multiphoton imaging of taste bud
structures
from tissue loaded with calcium sensitive dye, in this case calcium green
dextran.
Example 4 -- Electron Microscopy and Fixation Protocols
The inventors obtained high resolution scanning (SEM) and transmission
electron (TEM) micrographs in order to determine the ultrastructure of the pig
taste
bud and surrounding tissue. This aided in the interpretation of the stimulus-
response
data to be obtained from the MP-FLIM system. Fixation of biological tissue and
preparation for electron microscopy is complicated, so work began with a
general
mammalian fixative cocktail applied to small pieces of freshly obtained
tissue. Each
fixation trial used tissue for processing and imaging via light microscopy and
electron (scanning and transmission) microscopy.
Initial inspection of the fixation results via the surface morphology of the
papillae was performed by SEM. In most cases, a papilla from side of the
tongue
was used due to the slightly larger size of the papillae and the relative
abundance of
taste buds located in each. Many taste pores were observed scattered over the
papilla surface and were noted by the morphology and topographic form. Also
noted were the grooves discussed in literature as possibly being associated
with
assistance in guiding tastants to the taste pores. In several cases, multiple
taste pores
were noted within these grooves. In other cases, taste pores were noted as a
depressed or raised structure. It was noted during SEM observations at higher
magnification that few, if any, taste pores were located laterally to the
papilla. Most
taste pores are scattered across papillae dorsal surfaces.
It was difficult to develop a fixation protocol that would achieve acceptable
preservation of the taste cells, supporting cells, and nerve processes when
the test
tissue was obtained from an abattoir. A general mammalian taste bud fixative
from
literature was attempted initially. The fixative cocktail was made up of some
or all
of the following in distilled water: paraformaldehyde, glutaraldehyde, sodium
cacodylate, and sucrose. Modifications to the fixative concentrations and
components were made in subsequent trials in order to improve the tissue
quality
under light microscope observation. In no case, with adult tissue, did the
changes to
the fixative cocktail result in properly preserved tissue. The tissue
exhibited
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significant degradation, with gaps between cells and between the cytoplasm and
nuclei of taste cells. In addition, the damage appeared, upon close
inspection, to be
pre-fixative in nature. Three trials were performed on the adult tissue, with
variations in fixative composition and concentrations.
Subsequently, infant pig tissue from research pigs was obtained, and
subjected to the fixative cocktail developed for the adult, killed pig tissue.
This
resulted in acceptable tissue preservation for TEM and yielded the first
useful TEM
images of porcine taste tissue. The infant tissue showed much better
preservation,
with no large gaps or voids between cells or cytoplasm and nuclei. The
discovery
that the tissue obtained from killed pigs is generally degraded prior to
fixation
indicated that the tissue may not be viable for a suitable period after
slaughter.
There are likely many reasons for this, including inflammation due to stress
induced
at slaughter, feeding and other care deficiencies, or time between slaughter
and
removal of the tongue.
Example 5 -- lontophoretic Loading of Taste Tissue with Calcium Green-1
Dextran
The inventors assessed the efficacy of the iontophoretic loading method for
porcine taste tissue. Literature reports indicated successful loading of
dextran
conjugate dyes into rodent taste tissue (foliate papillae), but it was unclear
if the
process would be successful for pig fungiform papillae.
The fungiform papilla exhibited numerous pore structures on the surface of the
tissue that were somewhat visible under moderate magnification using a
specimen
preparation epifluorescence stereomicroscope. Initial experiments were
undertaken
to optimize the loading of individual taste pores using a variety of glass
pipette
configurations and tip sizes for iontophoresis. Optimized loading parameters
for
individual pores were found to utilize Corning 7740 borosilicate glass (1.5 mm
O.D.), pulled to a tip diameter of 4-6 m, resulting in the most specific
filling of
taste cells and the least amount of damage to the tissue. This was
accomplished for
one pore at a time with 1-mmol/L dye in deionized water, 4-6 microamperes of
current for 8-10 minutes.
In an effort to streamline the dye loading process, the inventors attempted to
simultaneously load many taste buds in a single papilla using a fire-polished
pipette
with a tip just large enough to fit over a single fungiform papilla (0.8-1.5
mm). The
initial experiments to load with a wide-bore capillary were successful in
loading all
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accessible taste buds in subject taste papillae reproducibly. Again, the
optimization
procedure was applied using the wide-bore capillary. Optimal loading
parameters
were found to utilize 5-mmol/L dye, 200-300 microamperes of current, for 15
minutes of loading time.
The loading procedures were developed utilizing large (30 mm x 15 mm)
sections of lingual tissue excised from the tongues with minimal muscle and
connective tissue retained on the tissue block. The first images obtained from
the
microscopic imaging system were captured from tissue immobilized onto a
coverslip
using tissue cement in a static pool of electrophysiological recording
solution or
artificial saliva. As the imaging system produces confocal images, three-
dimensional models obtained for z-series image stacks were prepared from
various
loaded papilla under various conditions. Variability in tissue quality yielded
mixed
results in terms of number of cells per bud that loaded, and the number of
buds
loaded per papilla. In some cases, the presence of pores on the papilla
surface was
not an indicator of the presence of loaded taste buds under the pores, after
iontophoretic loading. In these cases, the epithelial tissue surrounding the
pore
would load with dye but no fluorescence would originate from under the pore,
indicating that either no taste buds were present or the apical ends of the
taste cells
did not extend into the pore, and thus the taste cells did not load with dye.
Post-load taste bud visualization was performed using a Leica TCS confocal
microscopy system, and later using the visible laser line on the MP-FLIM
system.
Tissue was loaded with dye, one pore at a time, and then placed into an
imaging
chamber and imaged using the 488-nm excitation laser line and standard
confocal
optics in a z-series acquisition mode in order to generate an optically
sectioned
image stack for 3-D reconstruction. The images were maximum in plane
projections, or three-dimensional reconstructions of the z-series data.
Epithelial
nuclei were clearly visible and appeared to have preferentially taken up dye,
relative
to dye uptake in the cytoplasm. Alternatively, the nuclei may contain
significant
unbound calcium ion, resulting in enhanced emission. The imaged cells were
approximately 30-40 micrometers below the surface of the squamous epithelium
and
represent the practical limit for the interrogation depth by visible
microscopy (1-
photon excitation of the dye).
Upon complete installation of the MP-FLIM system the loading procedures
were evaluated using the system in 2-photon excitation mode. The detector
system
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was flexible enough that evaluations can be made using the descanned optical
detectors, or through a higher efficiency non-descanned detector system.
Initial
multiphoton spectroscopic scans were performed with calcium green dextran dye
solutions in order to determine maximum excitation efficiency for the dye
under 2-
photon excitation. Several high-resolution 3-dimensional datasets were
collected
over the course of the development work in order to provide insight into the
loading
efficiency and the types of cells that loaded. The data-sets were collected
using the
confocal descanned detector with a spectral window of 500-700 nm, pinhole open
to
600 micrometers, 810 nm laser wavelength at 1.70 W indicated power, with 512 x
512 or 1024 x 1024 image sizes. The number of images collected per loaded bud
depended on the level of dye loading to the basolateral membranes, but in
general,
50-70 micrometers of total depth was imaged at 0.3 to 0.5 micrometers per z-
slice.
The apical and basolateral processes were clearly visible extending from the
cell
body. The differences in number of cells loaded between tissue samples may be
related to any number of factors, including: tissue age, quality,
developmental state
of the papilla, and preferential loading of cell type.
Example 6-- Development of Stimulus Delivery System and Imaging
Protocols
Imaging, Tissue Chamber, and Stimulus Delivery Systems
Initial characterization of the tissue loading and morphology was performed
with pieces of tissue glued or cemented to a coverslip in an
electrophysiological
recording chamber. This approach was utilized with 40X and 63X water dipping
objective lenses with laser power and wavelength optimized for the calcium
green
dextran. The mounting procedure was sufficient for evaluation of loading but
did
not provide a means for quantitative control of stimulus delivery and removal.
A commercially available temperature controlled isolated taste epithelium
chamber was procured and utilized for mounting of an excised, dye loaded
tissue
block (5 mm x 5 mm). This allowed isolation of the apical perfusion of
stimulus
delivery medium and the oxygenated basolateral perfusion medium. Initially,
the
chamber was used in the open configuration. The tissue was placed between the
top
plate and bottom plate of the chamber, forming a membrane between two separate
perfusion baths. The chamber was sealed with high viscosity vacuum grease. The
bottom perfusion of oxygenated Tyrode's solution was achieved with a
peristaltic
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pump, initially, but this resulted in unacceptable pulsing of the tissue due
to the
pressure pulses induced from the peristaltic. The upper, open chamber was
continuously perfused with artificial saliva via a gravity fed system. A
constant
volume was maintained in this chamber by removing solution with a syringe
needle
fixed at the appropriate level and attached to a vacuum line. An aliquot of
stimulus
was applied to the upper chamber from one of five 30 cc syringe barrels
attached to
a manifold via independently activated pinch valves. These reservoirs are
pressurized if necessary. The system temperature is controlled to 30 degrees
Celsius
during stimulus experiments.
Due to control difficulties with the gravity feed liquid delivery system and
peristaltic pump, modifications were made to the chamber design and pump
system
in order to confine the flow path, reduce the volume of perfusate in contact
with the
tissue, reduce pump noise, and reduce plugging in the chamber inlet and outlet
lines.
A push-pull pumping scheme for perfusates was designed and implemented for
both
perfusion chambers using dual Harvard Apparatus PhD syringe pumps and glass
Gas-Tight syringes. These changes, coupled with Teflon tubing and finger-tight
high performance liquid chromatography couplers, resulted in reproducible flow
at
relatively low rates (0.05 mL/min. to 1.00 mL/min) with minimal pressure pulse
artifacts in the liquid stream. In addition, a remote controlled sample
injection valve
was ordered with variable injection loops available for introduction of
stimulus into
the fluidic system without reliance on gravity feed and pinch valves. Prior to
receipt
of the automated sample valve, a manual fixed volume (1.0 mL) valve was used
to
inject sample.
The flow characteristics of the system were measured using varying flow
rates of upper perfusion with fluorescein visible dye in basic solution (pH =
9). The
dye was injected into the sample loop and time series intensity acquisitions
made
using the 2-photon mode in order to visualize the dye introduction and removal
to
and from the sample chamber close to the tissue position. The data were
collected
using the xzt mode of the microscope with the injection loop switched to
"inject"
mode at the onset of data acquisition. Because the dipping objective creates
non-
laminar flow regimes around the tissue, the profiles are not as uniform as
would be
expected. Likewise, variability in the bead size surrounding the objective was
noted
during flow experiments. However, the sample injection loop and syringe pump
system provided improved sample injection integrity and flow stability
relative to
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the gravity feed system. However, non-uniform flow characteristics of the open
chamber coupled with the varying liquid bead size in contact with the
objective lens
resulted in irreproducible sample injection times as a function of flow rate.
In
addition, visual observation of the dye entrance, diffusion, and exit from the
chamber showed that a preferred flow path in the chamber was around the
objective
close to the surface of the open bath. Therefore, the stimulus flow rate is
necessarily
coupled to diffusion in terms of stimulus exposure to the tissue. This
coupling and
bead variability precludes the accurate prediction of the arrival time of
stimulus to
the tissue surface.
Instability in the tissue during imaging experiments while using the flowing
system led to a prolonged troubleshooting period where significant
improvements
were made in the tissue chamber design and the flow system as well as the
mechanical stability of the microscope. It was apparent once the flow
experiments
were begun that the manual stage did not provide the mechanical stability
required
that would prevent movement of the stage once tubing and electrical
connections
were made to the epithelial chamber. Under static conditions, the stage was
stable
enough to allow for high-resolution three-dimensional imaging. However, the
addition of fluid flow and variable mechanical load to the stage resulted in
significant movement during stimulus and acquisition of image data. In some
cases,
the tissue would move 20-30 micrometers in 60 seconds, rendering any confocal
imaging attempt useless. In order to stabilize the system, a computer
controlled x-y
stage was retrofitted to the microscope. This upgrade improved the stability
of the
system and allowed for continued troubleshooting of the residual movement
issues
that were attributable to flow instabilities and tissue mechanical response to
stimuli.
The most recent series of tissue mounting and flow system modifications
involved the conversion of the chamber to a gasket sealed closed chamber using
a
top coverslip, press-to-seal silicone gasket material, and a solid stainless
steel
pinhole substrate onto which the tissue was glued. The recent system
optimization
provided reproducible mounting and flow characteristics. The closed cell
provided a
uniform flow path, with no contact of the stimulus solution with the objective
lens,
thus providing additional stimulus timing stability as well as improving the
tissue
positional stability by removing the variable bead size atop the tissue in the
flow
cell. This improvement in the isolated epithelial chamber design improved the
function and allows a single papilla to be placed in the chamber with
reproducible
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mounting achievable. Finally, the closure of the flow cell results in the
trapping of
bubbles within the chamber. This results in compressible domains within the
flow
cell that produce pulses in the tissue position during switching of the sample
injection valve. An ultrasonic vacuum degassing system was installed to
provide
bubble free perfusate solutions and reduce the bubble entrainment in the
closed
system.
Example 7-- Standard Taste Stimuli Selection and Refractive Index Matching to
Perfusate
The choice of stimuli is driven by expectations for porcine response based on
limited literature information as well as by practical experimental
considerations. A
significant challenge was encountered upon initial stimulus application using
high
concentration stimuli. The stimulus solutions were prepared in artificial
saliva,
initially, and exhibited significant refractive index variability, based on
stimulus
concentration. For a stimulus such as sucrose, the concentration threshold is
high
enough for humans that the refractive index perturbation relative to blank
saliva is
on the order of 0.01 refractive index units. This refractive index mismatch
between
stimulus and perfusate resulted in significantaberration and focal plane shift
due to
the perturbation of the optical path during bath application of stimulus. As
the
optical path comprises the objective, perfusion bath, and tissue for intact
papilla
imaging using an upright microscopic configuration, this level of refractive
index
mismatch is intolerable. For a typical slice preparation, the introduction of
stimulus
is achieved via a countercurrent application of stimulus from a micropipette
positioned extremely close to the tissue and therefore stimulus does not
perturb the
imaging path. However, a slice preparation does not maintain the integrity of
the
taste bud and the polarization of the epithelium and for his reason we chose
the bath
stimulus system with intact papilla. It is probable that intensity-based
calcium-
imaging experiments reported in the literature that make use of bath stimulus
might
contain artifacts related to refractive index perturbations arising from
stimulus-
perfusion bath mismatch.
As the intact papilla is the preferred tissue sample type for this work,
refractive index matching of the stimulus and perfusion bath solutions was
pursued.
Taste-free, water soluble, low viscosity carboxymethylcellulose was procured
from
Sigma Aldrich in powder form to be used as a refractive index perturber.
Solutions
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of stimuli were prepared in artificial saliva, initially, and the refractive
index at 546
nm measured using a handheld refractometer. It was determined that a
refractive
index mismatch of 0.0003 refractive index units was sufficient to produce
focus
shifts and image distortions in the perfusion bath. Subsequently, a stock
solution of
CMC was prepared in deionized water or saliva having a refractive index on the
order of 1.3400. From dilutions of this stock solution, a calibration curve
was
developed relating CMC by weight in solution to the refractive index. Index
matched stimulus solutions were prepared using the CMC calibration and
adjusted
dropwise to within t0.0001 RI units of the target. In general, the most
concentrated
stimulus solution in a set of stimuli was chosen as the target for RI
matching. This
method was successfully applied to solutions prepared in deionized water and
in
artificial saliva and, in general, produced little to no refractive index
artifacts in the
image data.
Example 8-- Multiphoton Fluorescence Intensity and Lifetime-based
Response of Porcine Taste Tissue: Preliminary Lifetime Imaging of
Stimulated Tissue in Static Bath
Fresh porcine taste tissue was procured in the prescribed manner from a local
slaughterhouse and transported, after removal from the tongue, in chilled,
oxygenated Tyrode's solution to the preparation laboratory. The tissue was
affixed
to a coated Petri dish and taste buds iontophoretically loaded (1.0
microampere) with
calcium green dextran dye (1 mmol/L in DI water) via a drawn capillary
pipette.
Several taste pores were loaded on a papilla, with several papillae loaded
overall,
providing 3-5 taste buds loaded with dye. The tissue was mounted in a chamber
and
placed onto the microscope stage. The sample was imaged using the 63x water-
dipping objective, 60% laser power at 810-nm excitation, in xzy mode (256x256
pixels) in order to image the taste bud and surface features at once in a
longitudinal
section. The photomultiplier tube gain on the FLIM system was set to 85% and
the
initial Leica settings (non-optimized) were used to capture FLIM images of the
tissue over a 10-15 minute time course.
The same field of view was then imaged using the multiphoton lifetime
imaging capability of the instrument. The optical conditions for lifetime mage
collection are identical to intensity-based imaging, except that in photon
counting
mode the collection requires 30 seconds in this case for adequate signal to
noise.
The lifetimes were calculated by a double exponential fit of the decay curves
and
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color-coded based on average lifetime per pixel. Red values are shorter
lifetimes in
this case. The epithelial cells (surface cells), which load with dye, exhibit,
on
average, longer lifetimes than the taste cells located below the pore. The
shortest
lifetime evident in this image is associated with the apical end of the taste
cell
directly below the'pore. At rest, the average lifetime for all of the cells in
the field
of view is 1,634 picoseconds. The short lifetime component of the fit was set
to 350
picoseconds, an estimate of the short lifetime component based on initial
calcium
calibrations in order to facilitate rapid fitting of the data for comparison.
The taste bud was exposed to several gross stimuli: cycloheximide and
sucralose in deionized water. The purpose of this stimulus experiment was to
determine if the bud would remain viable during the time course of a stimulus
and if
possible, to determine the presence of a gross response via lifetime imaging.
The
stimulants were applied via bulk addition to the chamber holding the tissue:
0.5 mL
of 25-micromolar cycloheximide was first added after removal of 1 mL buffer;
0.5
mL of 1-millimolar sucralose added after the cycloheximide stimulus and
removal of
additional I mL buffer. The MP-FLIM image data were collected at approximately
1-minute intervals using the same optical and exposure conditions used to
generate
the resting image described above. Figure 9 shows the representative color-
coded
average lifetime images for the sequence of images captured during exposure to
the
different stimuli. The letters in the image correspond to subsequent sub-
images
collected during the experiment: a) rest; b) cycloheximide addition; e)
refocus f)
sucralose stimulus. Notice that the sucralose stimulus results in significant
shortening of the calcium green lifetime around the taste cells. As the
calcium green
lifetime associated with the epithelial cells remains essentially unchanged
during the
experiment, it is clear that he taste cells responded to the stimuli. It is
clear that the
calcium concentration surrounding the taste cells decreased during exposure to
the
sucralose stimulus, and in addition, color changes associated with calcium
green
lifetime changes are evident in cells not originally imaged in the cells at
rest.
Example 9 -- Multiphoton Fluorescence Intensity-based Response of Porcine
Taste Tissue: Preliminary Imaging of Stimulated Tissue in Flowing Perfusing
Medium
Tissue was received from the abattoir and loaded with calcium green-I
dextran using the optimized wide-bore loading method: 300 microamperes for 15
minutes. Two papillae in close proximity to one another were loaded in this
manner.
CA 02591959 2007-06-20
WO 2006/069142 PCT/US2005/046339
The papillae were extracted from the larger piece of tissue and placed into
the open
chamber configuration (this work was done prior to that using the closed,
gasketed
chamber). The tissue was repeatedly stimulated with 20 mmol/L citric acid
using
the 1.0 mL/min flow rate and 1.0 mL sample loop, switched to the load position
as
time series data acquisition began. The imaging parameters were as follows:
400 Hz
galvo scan rate, 2x zoom with 63x objective, 256x256 pixel image format, 2x
frame
average, 810 nm laser at 36%, and 0.5 Hz frame scan rate. The tissue was
received
at approximately 8:00 am, transported in oxygenated Tyrode's solution to the
lab,
loaded at approximately 10:00 am and imaged at approximately 11:00 am.
Cells from two adjacent taste buds within the second papilla were imaged in
xyzt mode at a depth of approximately 35 micrometers. Five cells from the
upper
taste bud and two cells from the lower bud appeared to load well with dye.
(See
figure 10). The time series data were processed using the alignment tools
development to address the tissue movement issues. An important point to be
made
here is the reproducible movement witnessed during numerous citric acid
stimuli.
The acid stimulated movement is not an artifact of the flow cell or due to
stage
instability; it is truly a mechanical response to a change in pH. However, the
movement in this case was primarily in the x-y plane, and therefore the
alignment
algorithm provided sufficient alignment.
It was noticed that several of the cells showed relative increases in average
intensity, indicative of an intracellular increase in calcium ion
concentration. In
most cases where movement was the dominant perturbation in the image data, the
movement is monotonic. However, it was clear that adjacent cells in the
examined
bud exhibit significant intensity changes corresponding to the acid stimulus.
Time series data show distinct stimulus-related changes in the relative
emission intensity for several cells. However, the relative difference in
emission
and differences in slope due to photobleaching makes comparison between cells
difficult. Therefore, a baseline correction was applied to the time series
data to
remove offset and slope differences between traces. The response decreased in
magnitude with subsequent stimulus, an indication that the tissue was reaching
the
end of viability (see figure 11). Given the time between slaughter and
exposure to
stimulus (4 hours) it is not surprising that the tissue appeared to be dead or
dying.
It was also noticed that other cells appeared to minimally respond to
successive stimuli with slight increases in average fluorescence intensity,
and other
51
CA 02591959 2007-06-20
WO 2006/069142 PCT/US2005/046339
cells in the system respond with a slight decrease in fluorescence intensity.
The
differential responses of different cells in the buds may be attributable to
either
intercellular trafficking of calcium ion, evidence of cell-to-cell
communication in
response to a single cell-signaling event due to taste receptors. Conversely,
taste
receptor cells may respond by either excitatory (increase in intracellular
calcium) or
inhibitory (decrease in intracellular calcium) activity.
All publications, patents and patent applications are incorporated herein by
reference. While in the foregoing specification this invention has been
described in
relation to certain preferred embodiments thereof, and many details have been
set
forth for purposes of illustration, it will be apparent to those skilled in
the art that the
invention is susceptible to additional embodiments and that certain of the
details
described herein may be varied considerably without departing from the basic
principles of the invention.
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