Note: Descriptions are shown in the official language in which they were submitted.
CA 02793840 2012-10-02
METHOD AND SYSTEM FOR OPTICAL MICROSCOPY
FIELD OF THE INVENTION
[001] The present invention relates to optical microscopy and more
particularly to providing
adjustable acceptance angle dark filed illumination in conjunction with
simultaneous
fluorescence imaging.
BACKGROUND OF THE INVENTION
[002] Optical microscopy involves passing light transmitted through or
reflected from the
sample through a single or multiple lenses to allow a magnified view of the
sample. The
resulting image can be detected directly by the eye, imaged on a photographic
plate or captured
digitally. The typical system of lenses and imaging equipment, along with the
appropriate
illumination equipment, sample stage and support, make up the optical
microscope. Typical
standard optical microscopy, bright field microscopy, suffers from limitations
which include that
it can only image dark or strongly refracting objects effectively, diffraction
limits resolution to
approximately 0.2 m in the visible region, and out of focus light from points
outside the focal
plane reduce image clarity.
[003] Optical microscopy of biological specimens, particularly live cells,
is difficult as they
generally lack sufficient contrast to be studied successfully; typically the
internal structures of
the cell are colourless and transparent. Commonly, contrast is increased by
staining the different
structures with selective dyes, but this involves killing and fixing the
sample. Staining may also
introduce artifacts; apparent structural details caused by the processing of
the specimen and are
thus not a legitimate feature of the specimen.
[004] Within the prior art these limitations have been overcome to some
extent by specific
microscopy techniques that can non-invasively increase the contrast of the
image. In general,
these techniques make use of differences in the refractive index of cell
structures. These include:
= Oblique illumination ¨ wherein side illumination gives the image a 3-
dimensional
appearance and can highlight otherwise invisible features;
= Dark field ¨ wherein directly transmitted light entering the image plane
is minimized
thereby collecting only the light scattered by the sample;
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CA 02793840 2012-10-02
= Dispersion staining ¨ wherein an optical technique results in a colored
image of a
colorless object, where five different microscope configurations are used
which include
brightfield Becke line, oblique, darkfield, phase contrast and objective stop
dispersion
staining;
= Phase contrast ¨ where differences in refractive index appear as
differences in contrast
within the image;
= Differential interference contrast ¨ also known as Nomarski contrast
microscopy wherein
differences in optical density appear as differences in relief an exploits
polarization
differences near refractive index boundaries;
= Interference reflection microscopy - used to examine the adhesion of
cells to a glass
surface, using polarized light of a narrow range of wavelengths to be
reflected whenever
there is an interface between two substances with different refractive
indices;
= Fluorescence - wherein certain compounds when illuminated with high
energy light emit
light of a different lower frequency and is of critical importance since it
can be extremely
sensitive allowing the detection of single molecules and wherein many
different
fluorescent dyes can be used to stain different structures or chemical
compounds
including one particularly powerful method being the combination of antibodies
coupled
to a fluorophore as in immunostaining;
= Confocal - wherein a scanning point of light instead of full sample
illumination is used to
give slightly higher resolution, and significant improvements in optical
sectioning;
= Single plane illumination microscopy and light sheet fluorescence
microscopy ¨ wherein
a plane of light formed by focusing light through a cylindrical lens at a
narrow angle or
by scanning a line of light in a plane perpendicular to the axis of objective,
allows high
resolution optical sections to be taken; and
= Deconvolution ¨ wherein the point spread function of the microscope
imaging system is
deconvolved by computer-based techniques in either two-dimensional or three-
dimensional domains.
[005]
There are also a multitude of super-resolution microscopy techniques to
circumvent
the diffraction barrier including for example serial time-encoded amplified
microscopy
(STEAM). These are typically based upon imaging a sufficiently static sample
multiple times
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CA 02793840 2012-10-02
and either modifying the excitation light or observing stochastic changes in
the image.
Additionally the knowledge of and chemical control of fluorophore photophysics
are at the core
of these techniques by which resolutions of approximately 20nm are attainable.
[006] Amongst the many biological systems of interest analysed with optical
microscopy is
the interaction between Actin and myosin, the two key contractile proteins in
muscle. Such
analyses have been studied for many years in the prior art using different
techniques. Amongst
such experiments in vitro Motility assays were extensively performed to obtain
new information
on the molecular mechanism of muscle contraction. Such assays take advantage
of the ability to
image rhodamine-phalloidin-labeled Actin filaments by fluorescence microscopy
as they interact
with and are translocated by myosin bound to a coverslip surface. In most
studies on single
Actin-myosin filament interactions, see for example Sellers in "In vitro
Motility Assays with
Actin" (Cell Biology Assays: Essential Methods, Ch. 20, Butterworth-Heinemann
2006), Jerry,
and Yamada, the two contractile filaments are not imaged simultaneously due to
technical
challenges. In some studies, however, Actin and myosin filaments were
visualized
simultaneously by either using fluorescence reagents/labels attached to both
Actin and myosin
filaments (Yamada) or by using the combination of dark field and fluorescent
microscopy
techniques, see for example Kalganov et al in "A Technique for Simultaneous
Measurement of
Force and Overlap between Single Muscle Filaments of Myosin and Actin"
(Biochemical and
Biophysical Research Communications, Vol. 403, pp351-356). Imaging both Actin
and myosin
filaments is important not only for visualization purposes but also for
measuring filament overlap
during active acto-myosin interactions because it should give new information
about cooperative
phenomena of myosin cross-bridges in myosin filaments.
[007] Myosin is known as the molecular motor which converts chemical energy
into
mechanical work. Thus any chemical reagents attached to myosin for imaging
purposes may or
may not change the ability of myosin to do its work. For this reason it is
critical to avoid using
fluorescent reagents conjugated with myosin when a study on Actin and myosin
interaction is to
be done. The inventors in their previous work, see Kalganov Rassier, showed a
technique where
fluorescent labeling of myosin is not required for simultaneous imaging and
force measurement.
In that work a standard Nikon immersion dark field condenser was used to
create dark field
images of myosin filaments. The disadvantage of using the dark field condenser
was in necessity
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CA 02793840 2012-10-02
to limit substantially the numerical aperture (NA) of the objective to form
the dark field image.
The objective's low NA caused Actin filaments to appear dark, hardly
distinguishable from the
background.
[008] Within the prior art Vodyanoy et al in US Patent 7,688,505 entitled
"Simultaneous
Observation of Darkfield Images and Fluorescence using Filter and Diaphragm"
teach to a
system employing an annular diaphragm and optical filter which are used for
simultaneous
observation of darkfield images and fluorescence. The diaphragm provides a
variable diameter
controlled by a lever and a removable filter which is used to adjust the
amount of unfiltered
incident light which produces the darkfield images when directed on a sample
whilst the
removable filter is used to filter light of the particular frequency for
producing fluorescence
images. However, Vodyanoy does not address the issues identified and discussed
supra in
respect of the NA of the optical system nor the requirement to use fluorescent
reagents.
[009] Accordingly it would be beneficial to provide an imaging technique
which would
allow simultaneous visualization of single Actin and myosin filaments as well
as the filament
overlap without requiring fluorescent conjugates for myosin filament
visualization. It would be
further beneficial for the imaging technique to improve, i.e. increasing,
(Actin) filament image
brightness contrast and signal-to-noise ratio (SNR).
[0010] Other aspects and features of the present invention will become
apparent to those
ordinarily skilled in the art upon review of the following description of
specific embodiments of
the invention in conjunction with the accompanying figures.
SUMMARY OF THE INVENTION
[0011] It is an object of the present invention to mitigate drawbacks of
the prior art with
respect to optical microscopy and more particularly to providing adjustable
acceptance angle
dark filed illumination in conjunction with simultaneous fluorescence imaging.
[0012] In accordance with an embodiment of the invention there is provided a
method
comprising:
providing a sample mount forming a predetermined portion of a visual
inspection system;
providing an imaging system comprising at least a lens for imaging a sample
upon the sample
mount;
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CA 02793840 2012-10-02
providing an illumination system for coupling light to a predetermined region
of the sample
mount imageable with the imaging system, wherein
the illumination system only illuminates the predetermined region of the
sample with light
incident exceeding a predetermined angle to the axis of the imaging system.
[0013] In accordance with an embodiment of the invention there is provided a
system
comprising:
a sample mount forming a predetermined portion of a visual inspection system;
an imaging system comprising at least a lens for imaging a sample upon the
sample mount;
an illumination system for coupling light to a predetermined region of the
sample mount
imageable with the imaging system, wherein
the illumination system only illuminates the predetermined region of the
sample with light
incident exceeding a predetermined angle to the axis of the imaging system.
[0014] In accordance with an embodiment of the invention there is provided a
system
comprising:
a plurality of optical emitters disposed at approximately constant radius from
a location to be
imaged;
an annular lens receiving the optical output from each optical emitter of the
plurality of optical
emitters and coupling these optical outputs to the location to be imaged;
an adjustable shield disposed on the inner edge of the annular lens, the
adjustable shield varying
the angular range of optical signals from the plurality of optical emitters
coupled to the
location to be imaged.
[0015] Other aspects and features of the present invention will become
apparent to those
ordinarily skilled in the art upon review of the following description of
specific embodiments of
the invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Embodiments of the present invention will now be described, by way of
example only,
with reference to the attached Figures, wherein:
[0017] Figure 1A depicts a commercial opto-mechanical assembly providing a
white short arc
light source coupled to a liquid optical waveguide and a reflective collimator
system according to
CA 02793840 2012-10-02
an embodiment of the invention to couple the liquid optical waveguide to a
multimode silica
optical waveguide for subsequent coupling to an optical microscopy test system
according to an
embodiment of the invention;
[0018] Figure 1B depicts an opto-mechanical assembly providing a reflective
collimator
system according to an embodiment of the invention such as depicted in Figure
1A to couple a
liquid optical waveguide to a multimode silica optical waveguide;
[0019] Figure 1C depicts a schematic presenting an effective point light
source model
exploited to design a reflective collimator system according to an embodiment
of the invention
such as described in Figures 1 A and 1B to couple a liquid optical waveguide
to a multimode
silica optical waveguide;
[0020] Figure 2A depicts a schematic of an illuminating chamber and optically
accessible
experimental bath according to an embodiment of the invention;
[0021] Figure 2B depicts an optical micrograph of an illuminating chamber and
optically
accessible experimental bath according to an embodiment of the invention with
multimode silica
optical waveguide illumination;
[0022] Figure 2C depicts an schematic of an illuminating chamber and optically
accessible
experimental bath according to an embodiment of the invention with electrical
excitation /
biological restraint elements in place;
[0023] Figure 2D depicts a holder for an optically accessible experimental
bath according to
an embodiment of the invention;
[0024] Figure 2E and 2F depict top and bottom elevation views of an
illuminating chamber
and optically accessible experimental bath according to an embodiment of the
invention;
[0025] Figure 3A depicts a cross-section three-dimensional schematic of an
illuminating
chamber and optically accessible experimental bath according to an embodiment
of the invention
with multimode silica optical waveguide illumination;
[0026] Figure 3B depicts a holder for an optically accessible experimental
bath according to
an embodiment of the invention as discrete element with optical coupling
elements and
assembled;
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[0027] Figures 4A through 41 depict optical micrographs of two Thick filaments
isolated from
muscles imaged with an optical illumination assembly according to an
embodiment of the
invention wherein Thick filaments become brighter the intensity of
illumination is increased;
[0028] Figure 5A and 5B depict a visual comparison of Thick muscle filament
appearance
imaged with a prior an dark field condenser system and an optical illumination
assembly
according to an embodiment of the invention respectively;
[0029] Figures 6A and 6B depict simultaneous imaging of single Thick and Actin
muscle
filaments using prior art dark field condenser;
[0030] Figures 6C through 6E depict simultaneous imaging of single Thick and
Actin muscle
filaments using an optical illumination assembly according to an embodiment of
the invention;
[0031] Figures 7A depict simultaneous imaging of single Thick and Actin muscle
filaments
using prior art dark field condenser;
[0032] Figure 7B and 7C depicts simultaneous imaging of single Thick and Actin
muscle
filaments using an optical illumination assembly according to an embodiment of
the invention;
[0033] Figures 8A through 8L depict in a sequence of video frames the active
interaction
between a single Actin and Thick muscle filament using an optical illumination
assembly
according to an embodiment of the invention;
[0034] Figures 9A through 9L depict color representations of the same video
frames shown in
Figures 8A through 8L depicting the active interaction between a single Actin
and Thick muscle
filament using an optical illumination assembly according to an embodiment of
the invention;
[0035] Figures 10A through 10L depicts the same video frame sequence as
Figures 8A
through 8L after background image subtraction to increased uniformity of the
Thick filament
intensity profile;
[0036] Figures 11A through 11L depicts color representations of the background
corrected
images presented in Figures 10A through 10L respectively;
[0037] Figures 12A through 12F depict optical micrographs of another
experiment where
single Actin and Thick filament interact as visualized using an optical
illumination assembly
according to an embodiment of the invention;
[0038] Figures 12G through 12L represent color representations of the images
presented in
Figures 12A through 12F respectively;
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CA 02793840 2012-10-02
[0039] Figures 13A through 13F depict color representation of the gray scale
video frames
depicted in Figures 12A through 12F respectively;
[0040] Figures 14A through 14C depicts a background image subtraction
procedure
exploiting data captured with an optical illumination assembly according to an
embodiment of
the invention wherein Figure 14B is subtracted from Figure 14A to yield Figure
14C;
[0041] Figure 15 depicts an optical illumination assembly according to an
embodiment of the
invention exploiting multiple excitation elements within a concept coined as a
"molecular
stadium"; and
[0042] Figure 16 depicts a "molecular stadium" according to an embodiment of
the invention.
DETAILED DESCRIPTION
[0043] The present invention is directed to optical microscopy and more
particularly to
providing adjustable acceptance angle dark filed illumination in conjunction
with simultaneous
fluorescence imaging.
[0044] The ensuing description provides exemplary embodiment(s) only, and is
not intended
to limit the scope, applicability or configuration of the disclosure. Rather,
the ensuing description
of the exemplary embodiment(s) will provide those skilled in the art with an
enabling description
for implementing an exemplary embodiment. It being understood that various
changes may be
made in the function and arrangement of elements without departing from the
spirit and scope as
set forth in the appended claims.
[0045] Within microscopy a lot of attention is normally paid to what
magnification and
resolution of objective was employed in the image acquisition but the very
important aspect of
how the sample was illuminated is usually dismissed. In order to develop an
imaging technique
to simultaneously visualize single Actin and myosin filament the inventors
considered
combining fluorescent microscopy and dark field microcopy, i.e. to use light
scattered as well as
that reflected and refracted from the sample in order to form the image of the
object being
imaged. However, conventional dark field microscopy does not work well in this
arrangement as
it requires the numerical aperture (NA) of the imaging objective be
restricted. As presented
below in respect of Equation (1) the NA of a lens defines the angular cone of
light that can be
accepted or exit the lens.
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CA 02793840 2012-10-02
NA = n = sin(0) (1)
where n is the refractive index within which the lens is working, e.g. air n
=1.00 , and /9 is the
half-angle of the maximum cone of light that can enter or exit the lens.
[0046] For a typical optical microscopy system when forming dark field images
the
NA 0.8, or 0 530, which leads to very poor quality for simultaneous
fluorescent imaging.
Imaging fluorescently labeled Actin filaments requires high objective NA,
typically in the range
of NA 1.2 ¨1.4 . As evident from Equation (1) such high NA's can only be
achieved with
immersion of the lens into a medium with n> 1.0, i.e. an oil with n 1.5. This
high NA allows
for the image to be bright, as the Actin filament brightness is proportional
to NA4, and the
objective works as an objective and a condenser at the same time. Limiting the
objective's NA
for dark field imaging is necessary because the angle at which the sample is
illuminated with the
condenser lens has to be higher than the angle of acceptance of the objective
so that light is
unable to come directly into the objective. When an oil immersion high NA
objective (1.2-1.4
NA) is used it becomes theoretically impossible to outperform its acceptance
angle if a dark field
condenser is immersed in a water solution which has lower refractive index
than the oil
nwaõ, =1333 , nod = 1.515).
[0047] This is a common problem for any applications where biological samples
are to be
imaged as they typically require water solutions to perform the experiments
within. One potential
solution to the problem is to add oil to the experimental solution to increase
its refractive index
and therefore the angle of illumination. However, such oil-water emulsions and
/ or solutions
will impact the biological system being imaged and analysed. Accordingly in
order to overcome
this limitation within the prior art the inventors undertook to design an
illumination system such
that the light would be coupled onto the sample at higher angles than the
objective could accept
such that only scattered light from the sample would be used by the objective
to form an image.
As noted above during dark field imaging the objective's NA had to be 0.8,
which for oil
immersion using an oil with n 1.52, defines an acceptance angle of 0 = 31.76 .
Accordingly,
an optical imaging system according to an embodiment of the invention for
simultaneous
fluorescent imaging and dark field imaging it is necessary to illuminate the
sample at a higher
angle than 0 = 31.76 in order to be able to increase the NA of the objective
above 0.8. For an
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CA 02793840 2012-10-02
NA=1.4 objective using an oil with n=--- 1.52 its angle of acceptance is
calculated to be
0=67.08 . Based on such considerations the inventors according to embodiments
of the
invention have designed and implemented an illumination system with a
continuously adjustable
angle of illumination of 65 90
allowing use of the full objective aperture and also
allowing determination of the angle at which the samples appear brightest or
highest contrast.
[0048] Designing and Engineering the Illumination System: A first
consideration in
respect of the dual function microscopy system according to an embodiment of
the invention is
the focusing of the light from a white light source into a multimode optical
fiber in order to
couple it into the experimental chamber. A large core (1mm) low NA (0.22)
fused silica high OH
optical fiber was selected. Whilst a lager fiber core allows for the
collection of more light from
the white light source a lower NA defines a lower exit angle of the light beam
from the fiber
which reduces the complexity of collimation allowing a smaller lens assembly.
The fiber was
chosen due to the low attenuation in the working wavelength region of the
biological
experiments, this being 500nm
600nm . An illumination system of four optical fibers to
bring light to the sample from four different sides was adopted to provide
symmetrical
illumination.
[0049] The fused silica optical fibers were jacketed and connectorized with
SMA905
connectors at one end and with smaller ST connectors at the other end. Each
optical fiber was
coupled to a different optical source, these being one 250W halogen light
source (ARC-TS-428
from Princeton Instruments) and three 120W electric arc light sources such as
normally used to
excite fluorescence in microscopy (X-Cite Series 120 from EXFO Lumen
Dinamics). These
electric arc light sources 100A, as depicted in Figure 1A, are supplied with
1.5m long liquid light
guide 100B having a 3mm core diameter and a NA of 0.3 NA. Accordingly, an
optical coupler
100C was designed to couple the 3mm 0.3 NA liquid core fiber guide to the lmm
0.22 NA fused
silica fiber guide. The optical coupler 100C as depicted in Figure 1B the
light from the liquid
fiber guide was collimated with an aspheric anti-reflection coated lens 130 of
diameter 12mm
having a focal length of lOmm and NA 0.545 which is higher than the optical
guide NA of 0.3.
[0050] It
would be evident that collimating an optical guide is not done perfectly as
the core is
relatively large and the core accordingly acts as multiple sources originating
at different points
on the optical light guide tip and having a wide range of exit angles such
that the end of the
CA 02793840 2012-10-02
optical light guide face cannot be described precisely by the traditional
point light source model.
Notwithstanding this the inventors collimated the light from the liquid guide
using effective
point light source model as depicted in Figure 1C. In this model an imaginable
(virtual) point
light source, S, inside of the fiber located on the central fiber axis at a
certain distance d from
the fiber tip. This distance can be calculated taking into account the fiber
NA which describes the
maximum half exit angle 9 and the radius of the light guide, r. Equation (2)
defines this
geometric construction wherein 0 is the half exit angle and can be found as
given by Equation
(3).
¨r =tg(0) (2)
0= arcsin(NA) (3)
d= _______________________________________________________________ (4)
tg (arcs in (NA ))
[0051] Accordingly, for NA= 0.3 and r =1.5mm , and from Equation (4) from re-
arranging
Equation (3) d = 4.77mm . Accordingly, an effective point light source placed
at the focal point
of the lens will produce a collimated beam. Therefore the light guide tip
should be positioned at a
distance Fd ¨d from the lens surface where Fd is the lens focal distance.
Accordingly, the light
guide tip should be positioned at 10.00mm ¨ 4.77mm = 5.23mm away from the
surface of the
aspheric anti-reflection coated lens 130. After the aspheric anti-reflection
coated lens 130 the
collimated optical beam is coupled to a parabolic mirror reflective collimator
160, in one
embodiment an RCO8SMA-P01 from Thor Labs. This provides an 1 lmm clear
aperture for the
input collimated light and it's NA of 0.167 which is lower than the fused
silica fiber NA of 0.22
and therefore allows for low loss coupling to the fused silica fiber. This
parabolic mirror
reflective collimator 160 has a constant focal distance over a large
wavelength range and
therefore has neither chromatic nor spherical aberrations in contrast to
regular lenses. The other
end of the parabolic mirror reflective collimator 160 was fitted with a
multimode silica
waveguide coupling 170, namely an SMA905 optical connector.
[0052] The end of the parabolic mirror reflective collimator 160 towards
aspheric anti-
reflection coated lens 130 has a 0.5" diameter interface with external
threading compatible with
standard 0.5" diameter lens tubes from the same supplier. As a result a 2"
long, 0.5" diameter
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CA 02793840 2012-10-02
lens tube was employed to house and align the remaining optical elements
within the optical
coupler 100C. The collimating lens, aspheric anti-reflection coated lens 130,
is held by a pair of
retaining rings, not shown for clarity. A pair of small optics 5mm diameter
ring adapters 110
were installed in the lens tube at lOmm distance from each other and held with
additional
retaining rings, not shown for clarity. The liquid guide metal connector 120,
having 5mm
external diameter and length 20mm, was inserted in these ring adapters and in
this manner
aligned against the aspheric anti-reflection coated lens 130 with the desired
5.23mm separation.
To hold the liquid guide metal connector 120 tight a threaded hole was made in
the lens tube
wall at 22rnm away from the edge where the light guide connector entered and
an M3 screw
employed to tighten the assembly. A standard GG400 UV glass filter 150, 12.5mm
diameter and
2mm thick, was also inserted to block UV emissions as the arc light sources
produce a strong UV
radiation that can be dangerous for a user of the illumination system. This
GG400 UV glass filter
150 may placed between the aspheric anti-reflection coated lens 130 and the
parabolic mirror
reflective collimator 160 as shown in the Figure 1B or between the aspheric
anti-reflection
coated lens 130 and tip of the liquid guide metal connector 120.
[0053] The light from the 250W halogen light source was focused into its fused
silica optical
fiber with a different optical configuration. The 250W halogen light source
comprises an internal
mirror which focuses light from the halogen bulb into a rectangular 6x4mm
illumination zone.
The focal plane is located outside of the light source housing about 7mm away
as the mirror has
a long 147mm focal distance and 70mm diameter. Accordingly the exiting light
comes out with
relatively low divergence, ¨ 13.4 , such that it could be coupled directly
into a reflective
collimator, such as parabolic mirror reflective collimator 160, to focus the
light into the fiber. To
align the reflective collimator against the light source the collimator was
mounted in an optics
mount which was in turn mounted on a 0.5" diameter 2" long optical post which
was inserted in
a 0.5" diameter 2" long post holder and therein into a post holder base. To
block UV radiation
from this light source a GG400 UV glass filter may be placed inside of the
same optics mount
where the collimator is mounted.
[0054] Ray Transfer Matrix Analysis: Light exiting each optical fiber on the
sample end
requires focusing to a small spot in order to maximize the density of light
and hence illumination
intensity of the sample. The smaller the spot size the higher the irradiance
from the sample and
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CA 02793840 2012-10-02
the brighter the sample's appearance but on the other hand machining
tolerances put some
restrictions on how small the spot size should be. In the embodiments of the
invention described
below in respect of Figures 2 through 15 a spot size of less than lmm diameter
was considered
out of bounds as machining tolerances would otherwise mean that the focal
regions from the four
fibers will not overlap. It would be evident that other machining tolerances
may allow for smaller
overlap regions, or in other embodiments of the invention larger overlap
regions, to be
established.
[0055] Accordingly, it was decided to keep light spot diameter within the
range
1.0mm diameter 3.0mm . The light spot diameter may also vary according to the
intensity
settings of the light source. To focus light exiting each fused silica optical
fiber in such a spot
size a dual lens combination, a plano-convex thin spherical lens and a plano-
convex cylindrical
lens, was employed although other optical designs may be employed without
departing from the
scope of the invention. Design of this lens assembly was undertaken by ray
transfer matrix
analysis was performed wherein as the light travels from a light source and
passes through
different optical interfaces the light beam diameter, x, as well as its
divergence, 0, can be
calculated using matrix algebra. Although the fused silica optical fiber has a
circular spot it is
necessary to consider the more generic case of an elliptical light spot which
has half-axes,
x,, }, and divergences, i0õ p These two components are needed as one of them,
xõ will
experience focusing effect at the curved interface of the cylindrical lens and
the other one, xp ,
will not. Thus at the sample an elliptical instead of circular light spot with
the dimensions
ixõ xp 1, and divergences, 10õ } will be generated.
[0056] To
simplify slightly the calculations the same effective point light source model
to
approximate the behavior of light exiting the fiber tip, similar to above in
respect of the
consideration of designing the liquid light guide to fused silica coupling. In
this model we have a
virtual point light source located on the fiber optical axes inside of the
optical fiber. The light
emitted by this effective point light source has zero initial dimensions, Ix,
=0, xp = 0} and
travels inside of the fiber a certain distance dfiber = After the light exits
the fiber it travels certain
distance in air d air before it hits the plano-convex spherical thin lens.
After passing this lens the
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CA 02793840 2012-10-02
light travels again certain distance in air, da,r2 , before it hits the curved
interface of the
cylindrical lens where the xs and xi,, components of light have different
behavior, namely xõ.
gets focused but xp simply refracts like at a flat interface. Then, at the
rear flat interface of the
cylindrical lens both xs and xp components have a simple refraction which is
neglected in our
model due to small refractive index difference between lens glass and water.
Further, the light
travels a certain distance in water, d water , until it reaches the sample.
The matrices describing
behavior of light at optical interfaces are given below in Equations (5)
through (8).
(1
Propagation in constant index medium: (5)
0 1
(1 0 \
Refraction at a flat interface is:
0 (6)
n2
( 1 0
Refraction at a curved interface is: n1 ¨ n2 n1
(7)
R = n2 n2
f 1 0\
Thin Lens is: 1
¨ ¨i (8)
f
[0057] Knowing these matrices we can write two algebra matrix equations for
the txõ xpl
components of the light spot as given in Equations (9) and (10) below.
[Xs-1 1 1 u A glasslwater air n ¨ glass 1) 0 A 1 IA
air 2 1 0--i d air d ftharl 0 -
(9)
Leg] - R0 glass 1 0 0 1 0fiber
= n .5 o 1 ¨f 1 - o
- x -0 _ _
1[1 dan.21 11 dtur d fiber 0
n 1 (10)
P
9 [1 d glass lwater1 0 0 u ¨1 5 0 1 ¨ ¨ 1-o 1
Eifiber
_ _ f
- _
_ .
[0058] Here Ofiber is the maximum half exit angle of the fiber and can be
calculated as
fiber = arcsin(NAfther). Under an initial condition (xso = o, x,,0 = 0), as
the light starts from a
point light source. Other variables are refractive index of the lens glass
noass , radius of curvature
14
CA 02793840 2012-10-02
of the cylindrical lens R0, and focal distance of the thin spherical lens f.
As a first
approximation 9, and Op can be set to zero meaning parallel light coming onto
the sample and
this system of equations can be solved analytically relative to Ro and f for
1.0mm xõxp. 3.0mm allowing the lenses to be specified.
[0059] Collimating Adapters for Optical Fibers: In order to realize the
optical model
described supra initially collimating adapters for the fused silica optical
fibers were designed and
implemented using single spherical anti-reflection coated piano-convex lenses
of diameter
6.0mm and focal length lOmm. Their NA of 0.287 was chosen to be higher than
the fiber's NA
of 0.22. These lenses allowed compact design of the collimating adapters.
Threading was
implemented within the collimating adapters and on the ST connectors of the
optical fibers such
that the adapters could be screwed on the ST connectors. Thus by turning the
collimating
adapters we could move them forth and back along the ST connectors, i.e.
closer or further away
from the fiber tip, such that depending on the experimental requirements can
focus or collimate
the light beam exiting the fiber. Based on the optical model described in the
previous section the
threading on the ST connector and inside of the collimating adapter was
implemented to allow
the collimating lens to travel from 5mm to lOmm away from the fiber tip. It
would be evident
that other optical configurations would be possible without departing from the
scope of the
invention.
[0060] Illuminating Chamber: To allow continuous adjustments of the angle of
illumination
of the optical fibers a chamber was designed as depicted in respect of Figures
2A through 2H.
The illuminating chamber (ILUC) consists of two metal frames, each made in a
shape of a semi-
circle having a radius of 50 mm and being 16mm wide and 7.5 mm thick. These
frames are
connected to a 100mm diameter and 6nrim thick round metal base via two metal
rods, each 3mm
in diameter and 27mm long located on the opposite sides of the round base.
These metal rods
serve as an axis of rotation for the frames. In the center of the base of the
chamber a rectangular
recess of dimensions 22 x 50mm is provided, matching the dimensions of a
standard microscope
cover slip, allowing placement of a cover slip and an experimental bath
providing controlled
experimental environments.
CA 02793840 2012-10-02
[0061] A threaded hole in the surface of each frame was drilled and a threaded
knob was put
in each hole. Two circular fiber connector holders were made on the bottom
surface of each
frame as shown in Figures 2A through 2H respectively. Optical fiber connectors
were inserted in
these holders and fixed in the required orientations such that the light from
the four fused silica
optical fibers would fall onto the surface of the cover slip in the same
location at the centre of the
cover slip. By turning the knob, the frame which carries the optical fibers
would move up or
down around the metal rods thereby adjusting its angle of orientation relative
to the horizontal
plane. Thus the vertical angle at which the optical fibers illuminate the
center of the chamber
would be accordingly adjusted.
[0062] Optically Accessible Experimental Bath: A design requirement of the
illumination
technique was to provide high angle of illumination requiring that the walls
of the optically
accessible experimental bath (OPAXB) be transparent to visible light. In this
manner light can
travel from the collimating adapters to the sample through the walls which
would allow up to 90
degrees illumination angle. Accordingly, the experimental based was designed
based upon an
aluminum bath 45mm long, lOmm wide and lOmm high machined such that it had
four empty
rectangular windows each 9mm long and 6mm high on its two long sides. These
pairs of
windows on each side of the bath were separated by 2mm wide metal surface
which was used to
make lmm diameter holes to insert metal tubes providing solution flow during
experiments. To
permit optical coupling from the collimating adapters through the windows anti-
reflection coated
plano-convex cylindrical lenses were chosen, as defined from the preceding
design analysis. In
this instance lenses with a radius of curvature of 15.5mm and back focal
length 26.3mm were
employed. These lenses were custom cut in 8 equal pieces 6.0 0.1mm long and
8.75 0.1mm
high which were then aligned such that four pieces of the cylindrical lens
were in the windows of
the bath such that the curved surface of the lens was outside of the bath and
flat surface of the
lens was inside of the bath.
[0063] This cylindrical lens was made from N-BK7 glass having a refractive
index
n =1.519 at A = 550nm. If the OPAXB is empty, i.e. there is a glass-air
interface on the flat
surface of the cylindrical lens then due to the large refractive index
difference between air,
n = 1.0, and the lens glass, ng =1.519 , the light beam is going to refract at
the interface and
16
CA 02793840 2012-10-02
miss the center of the cover slip surface. However, when the experimental bath
is filled with the
water based experimental solution then the refractive index difference between
the water,
n = 1.333, and the lens glass is reduced significantly small and accordingly
the deflection of the
light beam will be negligible and the light from all four optical fibers will
impinge as intended at
the center of the cover slip surface. To allow the OPAXB to mount inside the
chamber four
magnets were glued into the four corners of the rectangular recess of the
chamber and
correspondingly in the corners on the bottom of the OPAXB.
[0064] Accordingly, the OPAXB and ILUC are depicted in Figures 2A through 2F,
3A and
3B respectively, wherein:
= Figure 2A depicts the ILUC and OPAXB according to an embodiment of the
invention;
= Figure 2B depicts an optical micrograph of the ILUC and OPAXB according
to an
embodiment of the invention with the fused silica optical fibers and
collimators assembled;
= Figure 2C depicts the ILUC and OPAXB according to an embodiment of the
invention
with electrical excitation / biological restraint elements in place;
= Figure 2D depicts a holder for an OPAXB according to an embodiment of the
invention;
= Figure 2E and 2F depict top and bottom elevation views of the ILUC and
OPAXB
according to an embodiment of the invention with a single fused silica optical
fiber and
collimator assembly;
= Figure 2G depicts a cross-section three-dimensional schematic of the ILUC
and OPAXB
according to an embodiment of the invention;
= Figure 2H depicts top and bottom views of the holder for an OPAXB
according to an
embodiment of the invention together with the optical coupling elements as
discrete
elements.
[0065] Microscope System: The experiments and tests reported below in respect
of Figures 4
through 14 were performed using a microscope system consisting of a Nikon
Eclipse TE 2000
Microscope, a Nikon Plan-Fluor 100X/0.5-1.3 oil immersion objective which is
suitable for
bright field, dark field and fluorescent microscopy measurements, a Nikon
immersion dark field
condenser 1.2-1.43 NA, and Rolera-MGi EMCCD video camera with a frame rate of
31 fps and
magnified pixel size of 150nm. Within this assembly in the filter chamber a
standard high
efficiency filter set was employed for the 450nm
490nm high efficiency exciter was used
17
CA 02793840 2012-10-02
and the standard emission filter was replaced with a custom designed high
efficiency
500nm A 600nm emission filter which allowed use of the strong A =550nm peak of
the arc
lamps for optical fiber illumination. Another arc light source of the same
model was used to
excite Alexa 488-phallodin fluorescent dye used as the fluorescent label for
the Actin filaments.
[0066] Brightness, Contrast and SNR Definitions: To evaluate the quality of
the images
obtained with the experimental configuration according to an embodiment of the
invention and
compare image quality for different imaging techniques three image parameters
were evaluated,
namely relative brightness, contrast and signal-to-noise ratio (SNR). These
image parameters can
have different definitions depending on field and application so for clarity
our definitions are
provided here.
[0067] During experiments with the illumination configuration and OPAXB
according to an
embodiment of the invention 8-bit gray scale images are acquired in audio-
video interleaved
(AVI) format. An 8-bit gray scale image means that the intensity of every
pixel of this image can
vary from 0 corresponding to totally black pixel to 255 corresponding to
totally white pixel.
Brightness of an object in this case is basically average intensity of pixels
of the object ranging
from 0 to 255 gray values. Based on this we define the relative brightness of
the object as
Br = 10¨ lb, where /0 is average 8-bit intensity of the pixels in the object
and lb is the average
8-bit intensity of the pixels in the background. Br basically shows the
brightness of the sample
relative to the background i.e. how bright the object would appear if
background level was 0.
Using relative brightness instead of just brightness allows comparing
different experiments with
different background levels.
[0068] Contrast, C, shows by how many times the object is brighter than the
background and
is accordingly defined as C = /0//, . SNR is defined by Equation (11) below
where o-0 is the
standard deviation of pixels in the object that describes the noise in the
object, and 0-1, is the
standard deviation of pixels in the background that describes the noise in the
background.
¨ ii,)
SNR= ____________________________________________________________ (11)
Al(0-02 + b2)
[0069] Image analysis to determine pixel intensity and pixel standard
deviation was
performed using the ImageJ program created at the National Institutes of
Health. Determining
18
CA 02793840 2012-10-02
Co within the exemplary application of Actin filaments is relatively straight-
forward as they are
uniform. For the thick filaments it is more problematic as they are not
uniform along their length
and accordingly, the central (brightest) portion of the thick filament was
employed wherein the
average brightness remains constant to within a predetermined range, for
example 10% . /0
however was measured for the whole thick filament. A polygon selection tool
within the
program was used to draw a closed line along the object's edge such that the
average intensity
and standard deviation of the pixels within the object may be obtained.
Similarly average
intensity of the background was obtained for two spots in the image. One
measurement was
taken next to the location of the object and the other measurement was taken
in the darkest
corner of the image. Both background values were used to calculate relative
brightness, contrast,
and SNR of the object. This approach with two background level measurements
allows
correcting for brightness gradient across the image in case the background is
not uniform and
obtaining more objective values of the image parameters.
[0070] Within this exemplary embodiment of the invention background brightness
was
measured using the average background value excluding those brightest regions
of the image
which are formed by other filaments, contaminations of solution, etc whether
in or out of focus.
Such artifacts are irrelevant to the measurements and may appear / disappear
in different
experiments. Accordingly, for consistency between experiments those regions
where other
filament and contaminants were present were excluded whether in or out of
focus.
[0071] Muscle Protein Preparation and Motility Experiments: Two muscle protein
filaments were used in the studies described below in respect of Figures 4
through 14. These
being Actin filaments and Thick filaments. Actin was purchased from
Cytoskeleton in powder
form before being re-suspended in a pH 7.0 Actin storage buffer solution
containing as active
elements 0.1 M potassium chloride (KC1), 4 mM Irnidazole, 2 mM magnesium
chloride
(MgC12), 1 mM sodium azide, 0.5 mM adenosine triphosphate (ATP), and 1 mM
dithiothreitol
(DTT)d. Under these conditions Actin self-polymerizes into filaments. Actin
filaments were
stained with Alexa Fluor 488 Phalloidin fluorescent dye according to a
standard procedure.
Native Thick filaments consisting primarily of myosin were isolated following
a standard
procedure from the anterior byssus retractor muscles of bivalve Mollusca
(mussels) and stored in
19
CA 02793840 2012-10-02
a pH 7.0 Thick filament buffer containing as active elements 10 mM piperazine-
N,N'-bis(2-
ethanesulfonic acid) (PIPES), 10 mM MgC12, 2 mM ethylene glycol tetraacetic
acid (EGTA),
and 2 mM DTT without any ATP.
[0072]
Motility experiments were performed using the following basic procedure with
slight
modifications. Initially, a standard 22 x 50mm cover slip was treated with
ethanol which was
allowed to evaporate before the cover slip was attached onto the bottom of the
OPAXB using
high vacuum grease. The OPAXB was installed in the recess of the ILUC and
retained using the
magnets. The ILUC with the optical fibers connected was then placed onto the
microscope table,
fixed tight with the ILUC retaining plates using M3 screws and centered
relative to the position
of the objective lens. Thick filaments were diluted by 1000 times in 5 ml of
AB/BSA/GOC/DTT/ATP with calcium (Ca) solution containing 25 mM Immidazol-HC1
(pH
7.4), 25 mM KC1, 4 mM MgC12, 1 mM EGTA, 1 mM DTT, 0.5 mg/ml Bovine serum
albumin
(BSA), 0.018 mg/ml Catalase, 0.1 mg/ml Glucose Oxidase, 3 mg/ml Glucose, 100
M ATP, 20
mM DTT, 2 mM calcium chloride (CaC12 ) and were put in the OPAXB to fill it
up. Such a large
dilution was employed so that it was possible to observe around one Thick
filament per field of
view in order to reduce the level of background and background noise which is
critical when
measurements of brightness contrast and SNR are to be taken. Thick filaments
were let to settle
for 5-10 minutes.
[0073] Next the optical fiber illumination was turned on and one or a couple
of Thick
filaments were detected. Angle of illumination, collimating adapters
positions, light sources
intensity were adjusted such that the brightness of the Thick filaments was
maximized and the
brightness of background minimized. Actin filaments were also diluted in
AB/BSA/GOC/DTT/ATP/Ca to 67 ng/ml and then 10 I of this solution was added
into the
OPAXB near the center of the cover slip where a Thick filament was located.
Fluorescent
excitation illumination was turned on and after approximately 1 to 5 minutes a
few Actin
filaments were observed in the field of view floating slightly above the cover
slip surface near
the Thick filament. Waiting for another 5-10 minutes was usually sufficient to
begin observation
of an Actin filament landing and sliding on the Thick filament which could
then be imaged
simultaneously using the optical microscopy system according to an embodiment
of the
CA 02793840 2012-10-02
invention. Images of the motion of Actin filaments simultaneously visualized
with Thick
filaments were recorded and stored on the computer for further analysis.
[0074] Comnarison between Prior Art Nikon Dark Field Condenser and New
Illumination Technique for Muscle Filament Imaging.
[0075] Simple Imaging: Initially the ability of the ILUC and optical
illumination system
according to an embodiment of the invention were evaluated for visualizing
Thick filaments.
Referring to first image set depicted in Figures 4A through 4E respectively
and second image set
depicted in Figures 4F through 41 respectively each image set depicts a Thick
filament imaged
with the ILUC. The intensity of the illumination light sources was increased
during these image
sets allowing control of the brightness of the filaments.
[0076] Subsequently, a few Thick filaments were imaged with a Nikon dark field
condenser
objective, Figure 5A, as well as with the ILUC according to an embodiment of
the invention,
Figure 5B. The imaging quality was compared subjectively, i.e. visually, as
well as objectively
using ImageJ analysis and measurements of relative brightness, contrast and
image SNR as
presented below in Table 1. It can be seen from the images in Figures 5A and
5B that
subjectively the appearance of Thick filaments is similar in both cases.
Similarly, relative
brightness, contrast and SNR values derived from the images likewise show no
significant
difference of imaging quality irrespective of whether the Thick filaments were
imaged with the
Nikon dark field condenser, i.e. NA 0.8, or with the ILUC, NA 1.2. These being
shown in first
and second lines of Table 1 identified as Thick - Dark and Thick - ILUC
respectively.
Relative Std. Dev. Contrast Std. Dev. SNR Std.
Dev.
Brightness Relative Average Contrast Average SNR
Average Brightness
Thick - 95.36 22.11 2.06 0.34 6.01 1.63
Dark
Thick - 108.54 31.52 2.93 1.13 7.34 1.68
ILUC
Simult. - 57.07 28.26 1.57 0.27 4.67 1.73
Dark =
Simult. - 71.38 20.69 1.93 0.43 5.60 1.11
ILUC
Actin - 13.37 2.25 1.17 0.065 1.51 0.43
Dark
Actin - 61.31 9.79 1.98 0.348 6.31 1.11
21
CA 02793840 2012-10-02
ILUC
Table 1: Experimental Results for Dark Field and ILUC Imaging (Note 1)
Thick ¨ Dark: Nikon dark field condenser lens, Thick filaments only, n=10;
Thick ¨ ILUC: ILUC according to embodiment of the invention, Thick
filaments only,
n=54;
Simult. ¨ Dark: Nikon dark field condenser lens, Thick filaments
simultaneously, n=34;
Simult. ¨ ILUC: ILUC according to embodiment of the invention, Thick
filaments
simultaneously, n=28;
Actin ¨ Dark: Nikon dark field condenser lens, Actin filaments
simultaneously, n=14;
Actin ¨ ILUC: ILUC according to embodiment of the invention, Actin
filaments
simultaneously, n=53.
(Note 1: Where the thick filament relative brightness was less than 50 gray
scale units these
thick filaments were not taken into account as they would not be used in real
experiment)
[0077] Next imaging tests were performed using both Actin and Thick filaments
simultaneously. Using the Nikon dark field condenser illumination the
objectives NA was again
0.8 and Thick filaments were clearly visualized whereas Actin filaments were
dark and hard to
distinguish in the background, see Figures 6A, 6B and 7A respectively.
Subsequently, using the
new illumination technique according to an embodiment of the invention the
objective's NA was
increased to 1.2, wherein Thick filaments were clearly imaged at equivalent
quality as with dark
field condenser but Actin filaments appeared much brighter as the result of
high objective's NA.
Typical images under this scenario being depicted in Figures 6C through 6E and
Figures 7B
through 7C respectively. Relative brightness, contrast and SNR values for
simultaneously
imaged Actin and Thick filaments with dark field condenser and the new
illumination technique
were calculated and summarized in the third to sixth lines of Table 1. From
these a significant
improvement of 4.57 times in relative brightness and 4.18 times in SNR of
Actin filaments using
the new illumination technique was observed. Some improvement of 1.68 times in
contrast has
also been obtained. Thick filament relative brightness, contrast and SNR
values were similar
irrespective of whether the dark field condenser or the new illumination
technique according to
an embodiment of the invention was used to visualize them in this simultaneous
imaging test.
22
CA 02793840 2012-10-02
[0078] Interaction and Sliding of Muscle Filaments Imaged with New
Illumination
Technique: Images shown and discussed in this section have been processed with
ImageJ
software by the application of a filter, e.g. mean filter of radius 1 pixel,
to smooth out noise.
Image brightness and contrast were improved with standard ImageJ tools as
well. These simple
image adjustments are normal to improve image quality in low light single
Actin filament
fluorescence (single molecule fluorescence applications) like ours where SNR
is low and
typically around 5. Motility experiments according to the procedure described
above were
undertaken. In this set of experiments a freely suspended Actin filament
spontaneously attaches
to a fixed Thick filament and moves along the Thick filament. Both Actin and
Thick filaments
were detected during an experiment and the motion of Actin filaments along
Thick filaments was
clearly visualized. Referring to Figures 8A through 8L processed gray scale
images of a sliding
experiment are depicted wherein the scale bar is 1000nm (ltim).
[0079] Figure 8A: Two Thick filaments are present in the image and indicated
with white
arrows.
[0080] Figure 8B: A freely suspended Actin filament came from above the cover
slip and
attached with one end to the Thick filament, the point of attachment is
indicated with a regular
arrow. The other end of the Actin filament indicated with an open arrow is not
yet attached to the
Thick filament
[0081] Figure 8C: The Actin filament continues to attach to the Thick
filament. The next
point of attachment is indicated with the regular arrow while free end of
Actin filament is
indicated with open arrow.
[0082] Figure 8D: The whole Actin filament is now attached to the Thick
filament.
[0083] Figures 8E through 8L: The Actin filament slides along the Thick
filament. The sliding
takes place from the Thick filament center and off the Thick filament. The
overlap between the
filaments is indicated with two regular arrows while free end of Actin
filament is indicated with
open arrow.
[0084]
Referring to Figures 9A through 9L respectively false color representations of
the
intensity profile of the images presented in respect of Figures 8A through 8L
respectively.
Conversion of the 2-D XY grayscale images into 3-D color images was performed
using ImageJ
software, through the Interactive 3D Surface Plot function. Accordingly the Z
axis in these
23
CA 02793840 2012-10-02
images represents the intensity profile of the image where different colors
indicate different
levels of intensity. The color legends in the Figures 9A through 9L
respectively show which
color corresponds to which level of gray scale intensity. In many instances
this color
representation of gray scale video data is more informative and makes the
filament appearance
more evident as well as filament overlap more distinguishable.
[0085] It can be seen from the images presented above in respect of Figures 8A
through 8L
and Figures 9A through 9L respectively that the intensity profile of a Thick
filament is not
always uniform. Frequently, a Thick filament will appear brighter in the
center and darker at the
ends. Under these circumstances as an Actin filament slides along the Thick
filament it may be
difficult to detect precisely where the Actin filament begins and where it
ends and so it is
difficult to detect the filament overlap precisely. Accordingly in order to
improve the ability to
distinguish both Actin and Thick filaments together with their overlap during
interaction the
inventors also generated a new image processing sequence, similar to
background subtraction, to
make the Thick filament intensity uniform all along its length. For this
purpose a single image of
Thick filament, such as depicted in Figure 14A, was taken, duplicated and an
offset of its
brightness by -100 gray scale units undertaken using ImageJ software. The
result of this
operation being shown in Figure 14B. Next, using image calculator command of
ImageJ the
offset image Figure 14B was subtracted from every frame of the same video
sequence where the
Thick filament and an Actin filament were interacting. As shown in Figure 14C
this procedure
lead to very uniform intensity profile of the Thick filament and as the result
during interaction
filament overlap intensity signal appears perfectly rectangular with sharp
edges.
[0086] Figures 10A through 10L depict the same video frame sequence as Figures
8A through
8L after background image according to the process described in respect of
Figure 14A through
14C in order to make the Thick filament intensity profile uniform. As such it
is evident where
both filaments begin and where they end such that during interaction it allows
more precise
determination of the filament overlap. Figures 11A through 11L depict the
color representation
of the same video frames shown in Figures 10A through 10L respectively. Both
Actin and Thick
filaments are clearly seen and filament overlap can be measured precisely.
[0087] Based upon these experimental procedures employing the illumination
system
according to an embodiment of the invention when the Actin filament fully
attached to the Thick
24
CA 02793840 2012-10-02
filament, see Figure 10D, the overlap was measured as 3098nm. This overlap
reduces as the
Actin filament slides off the Thick filament, as depicted sequentially in
Figures 10E through 10L
respectively wherein at the end of the experiment the filament overlap was
421m, see Figure
10L.
[0088] Figures 12A through 12F depict optical micrographs of another
experiment where
single Actin and Thick filaments interact as visualized using an optical
illumination assembly
according to an embodiment of the invention. Figures 120 through 12L represent
color
representations of the images presented in Figures 12A through 12F
respectively. Within the
experiment shown in the images of Figures 12A through 12F, and accordingly
also Figures 12G
through 12L, the filament overlap was 3360nm. This experiment is also depicted
in Figures 13A
through 13F respectively wherein color representation side view intensity
profiles are depicted.
In many instances measuring the filament overlap using side view images can be
more
convenient than in the top view images because intensity levels of filaments
and filament overlap
are indicated more clearly.
[0089] Within the descriptions of embodiments of the invention of the
illumination system in
respect of Figures 1A through 3 and the resulting experimental results using
the illumination
system according to embodiments of the invention a new technique allowing
simultaneous
bright, high contrast high SNR visualization of biological samples, in this
case two key
contractile muscle filaments, namely Actin and Thick filaments. Brightness and
SNR of Actin
filaments were significantly improved by 4.57 and 4.18 times respectively
compared to previous
known techniques in the prior art. Additionally, contrast of Actin filaments
has also been
improved by 1.68 times although Actin filament contrast values can vary
depending on how
bright the Thick filament illumination is. This arises as brighter Thick
filament illumination
causes higher background level of the image which lowers Actin filament (as
well as Thick
filament) contrast.
[0090]
Theoretically the brightness of Actin filaments is proportional to the
objective's NA to
the fourth power. Based on this how the brightness of Actin filaments should
increase when the
NA changes from 0.8, for the experiments using prior art dark field condenser,
to 1.2 for the
experiments using the fiber illumination technique according to an embodiment
of the invention.
Equation (12) provides the theoretical ratio of brightness with varying NA.
CA 02793840 2012-10-02
(Brightness ¨ OptkalFiber)4(1.2)4
= = 5.06 (12)
(Brightest ¨ DarkField)4 (0.8)4
[0091] Accordingly, the Actin filament brightness increases in an
illumination system
according to an embodiment of the invention by 5.06 times for these numerical
apertures which
is close to the experimentally obtained value of 4.57. In addition to the
image parameters being
significantly improved the technique according to an embodiment of the
invention allowed for
precise measurements of Thick and Actin filament overlap, one of the key
parameters in the field
of muscle biophysics. Within different experiments overlap values ranged from
about 400nm to
about 4000nm. Beneficially, the optical illumination system according to
embodiments of the
invention allows this simultaneous imaging technique to be applied to a wide
range of biological
systems. Any fluorescent and non-fluorescent objects can be imaged
simultaneously. Typically,
the non-fluorescent object would have a lower dimensional limit of
approximately 50nm in
diameter to provide sufficient image brightness although it would be evident
to one skilled in the
art that adjustments in the optical illumination design may adjust this.
[0092] Beneficially, unlike the prior art dark field condenser situation
the illumination
technique according to embodiments of the invention does not have illumination
system
elements on the top of the experimental chamber which could block access to
the sample being
observed, characterized and evaluated. This is a very important improvement
for applications
wherein active manipulation of the sample is part of the experimental
procedure. Within the
descriptions the OPAXB was designed and engineered so that it can be used
together with the
ILUC not only as a dark field illumination device but also as the highest
resolution bright field
illumination device. This becomes possible because the system allows the angle
of illumination
to be matched with the maximum angle of acceptance of the objective. This is
usually impossible
using standard bright field condensers in biological applications as a bright
field condenser is
immersed in a water solution which has lower refractive index, n =1.33 , than
objective oil,
typically n = 1.515, and therefore the highest maximum possible resolution
cannot be achieved.
[0093] Dark field imaging requires significant illumination to image small
objects as only
scattered light is used to build an image. This is the cost for the high
contrast allowed by dark
field imaging techniques compared to other imaging techniques such as bright
field. In many
biological systems, such as the muscle filaments employed supra which are
typically 50-90nm in
26
CA 02793840 2012-10-02
diameter these should scatter light according to Rayleigh scattering. In this
scenario the intensity
and therefore the brightness of the object is proportional to D6, where D is a
characteristic size
of the object, e.g. the Thick filament diameter. Accordingly, the brightness
of the filaments is
very sensitive to its diameter.
[0094] Accordingly to the embodiments of the invention described supra 3 or 4
light sources,
depending on the experiment, were connected to the lLUC via optical fibers.
Actually, imaging
of Thick filaments using only one optical fiber bringing light from only one
short arc or halogen
light source was demonstrated and multiple fibers were employed to provide
symmetrical
illumination. Accordingly, an alternative illumination system may provide
multiple fibers and
multiple sources to provide uniform excitation over wider optical illumination
power range.
Illuminating the samples from multiple angles increases the probability that
the light will scatter
from the sample at the right angle so that it will enter in the objective.
This is because scattering
angles will be different depending on the orientation of the sample in space
which is random for
every filament in every experiment. Accordingly, visualization of small
objects would be
improved through providing an infinite number of infinitely small light
sources illuminating the
sample from all possible aspects (angles) such that the probability that the
light scattered from a
randomly orientated filament will enter in the objective is maximized.
[0095] Accordingly referring to Figure 15 there is depicted another embodiment
of the
illumination concept and image visualization described above in respect of
Figures lA through
2H. As depicted in cross-section 1500A a microscope support 1510 has a
circular Lens
Assembly 1540 which includes a circular frame 1505 supporting a Cover Slip
1530 at its centre
and provides access for a Microscope Objective 1520 on the lower side. As
depicted in the
expanded cross-section, wherein the circular frame has been omitted for
clarity, a Circuit Board
1590 has mounted atop an Optical Sources 1580 which is coupled to an Annular
Lens 1570 that
couples the light emitted from the Optical Sources 1580 to the Sample 1550.
The Lens 1570
having an Adjustable Shield 1560 disposed on the inside of the annular disc.
The Adjustable
Shield 1560 allows the range of incident angles of the Optical Sources 1580 to
be adjusted
allowing the NA of the imaging objective can be similarly varied such that the
incident light is
not directly coupled into the imaging objective. Accordingly, as the NA is
increased the
Adjustable Shield 1560 is lowered towards the sample.
27
CA 02793840 2012-10-02
[0096] As evident from plan 1500B the Annular Lens 1570 is circularly
symmetric and sits
atop a plurality of Optical Sources 1580 which are provided in a circular
configuration such that
they are all coupled to the Sample 1550 via the circular Adjustable Shield
1560. In this manner a
large number of Optical Sources 1580 can be provided, such as discussed supra
in respect of
increasing the probability of scattering from the sample into the Microscope
Objective 1520. An
optional heatsink 1590 may be provided beneath the Optical Source 1580 and
Circuit Board
1590. It would be evident to one skilled in the art that Optical Sources 1580
may for example be
Light Emitting Diodes of one or more type. For example, these may be discrete
infrared, red,
yellow, green, blue, or ultraviolet LEDs or a combination of these.
Optionally, these may be
white LEDs exploiting ROB tri-chromatic assemblies, tetrachromatic assemblies,
or phosphors
for example. Alternatively, organic LEDs, active-matrix organic light-emitting
diodes
(AMOLEDs), incandescent sources, halogen sources, etc may be employed
discretely or in
combination with other sources.
[0097] It would also be evident that by appropriate design of the Annular Lens
1570 and
placement of Optical Sources 1580 that multiple annular rings of emitters may
be employed
including for example ultraviolet emitters for stimulating fluorescent
biological markers, white
LEDs and narrowband emitters of specific wavelength ranges. It would be
evident that other
designs may exploit annular lens sections rather than providing a complete
annular ring.
[0098] Referring to Figure 16 another embodiment of the invention with respect
to a
"molecular stadium" is presented. As depicted a Lens 1670 forms an annular
configuration
around a space within which the Sample 1650 is disposed. Disposed on the inner
ring of the Lens
1670 is an Adjustable Shield 1660, similar to Adjustable Shield 1560 in Figure
15. Disposed
around the Lens 1670 are a plurality of Optical Sources A and B 1680 and 1685
respectively that
are mounted to Circuit Board 1690 and heat Sink 1695. Lens 1670 is designed to
couple light
from each Optical Sources A and B 1680 and 1685 respectively to the Sample
1650 and is
designed in dependence of characteristics of the optical imaging system
including but not limited
to NA of image acquisition system and optical characteristics of Optical
Sources A and B 1680
and 1685 respectively such as beam divergence. Within the embodiments of the
invention
described in respect of Figures 15 and 16 the Adjustable Lens 1560 or 1660
respectively is
described in a configuration wherein motion in a direction parallel to an
optical axis of the
28
CA 02793840 2012-10-02
optical imaging system has been described. However, it would be evident to one
skilled in the art
that alternatively the motion may be in a direction at an angle to the optical
axis of the optical
imaging system, e.g. perpendicular, or may rotate relative to this optical
axis. Optical Sources A
and B 1680 and 1685 respectively may for example be ultraviolet LEDs and white
LEDs
respectively allowing fluorescent excitation as well as optical imaging. As
discussed supra
Optical Sources A and B 1680 and 1685 respectively may be selected from a wide
range of
optical sources according to the experiments being performed.
[0099] Within the embodiments of the invention described above in respect of
Figures 2A
through 16 the imaging has been described in terms of a microscope object that
forms part of the
imaging system providing simultaneous fluorescent imaging and dark field
imaging. However, it
would be apparent that other optical imaging designs may be employed according
to
embodiments of the invention without departing from the scope of the invention
and that the
design of such optical imaging designs in combination with design variations
to the illumination
systems and experimental chambers may allow combinations of other imaging
techniques to be
used simultaneously rather than sequentially upon a single specimen or upon
multiple specimens.
[00100] Further, in describing representative embodiments of the present
invention, the
specification may have presented the method and/or process of the present
invention as a
particular sequence of steps. However, to the extent that the method or
process does not rely on
the particular order of steps set forth herein, the method or process should
not be limited to the
particular sequence of steps described. As one of ordinary skill in the art
would appreciate, other
sequences of steps may be possible. Therefore, the particular order of the
steps set forth in the
specification should not be construed as limitations on the claims. In
addition, the claims directed
to the method and/or process of the present invention should not be limited to
the performance of
their steps in the order written, and one skilled in the art can readily
appreciate that the sequences
may be varied and still remain within the spirit and scope of the present
invention.
29
