Note: Descriptions are shown in the official language in which they were submitted.
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Virtual Fiducials
Cross-Reference to Related Applications
This application is a continuation-in-part of U.S. application
.. Ser. 16/658,052, filed October 19, 2019, entitled Microscope for Locating
Structures on the Inner Surface of a Fluidic Channel [SEQ-11]; and of U.S.
application Ser. 16/824,632, filed March 19, 2020, entitled Large-Field
Imaging
for Sequencing Instruments [CHE-211, which claims the benefit of priority to
U.S. provisional application 62/821,393, filed March 20, 2019, entitled Large
Field Imaging System [CHE-11p]; the entireties of all of which are
incorporated
herein by reference.
Field of the Invention
The present invention relates to a microscope. In particular, the invention
relates to a microscope for focusing on and locating structures at a partially
reflective interface where multiple partially reflective interfaces are
present.
Background of the Invention
In certain industries, such as genetic sequencing and genetic research, it is
desired to detect the nucleotides which are characteristic chemical moieties
of
nucleotides which constitute nucleic acids. Five nucleobases¨adenine (A),
cytosine (C), guanine (G), thymine (T), and uracil (U)¨are called primary or
canonical. They function as the fundamental units of the genetic code, with
the
bases A, G, C, and T being found in DNA while A, G, C, and U are found
in RNA. Rare bases have also been found in nature, such as 5-methylcytosine
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and other methylated bases, 5-hydroxymethylcytosine, 5-formylcytosine, and
5-carbosylcytosine. Other noncanonical bases include isoguanine, isocytosine,
and universal bases such as inosine.
These nucleotides can be detected using fluorescent labeling specific to
.. each type of nucleobase. The types of fluorescent labeling include direct
labeling by covalent labeling of nucleic acids with a fluorescent label or
noncovalent binding or intercalation of a fluorescent dye to nucleic acids,
and
indirect labeling via covalent attachment of a secondary label to a nucleic
acid,
and then binding this to a fluorescently labeled ligand binder. An alternative
indirect strategy involves binding of a nucleic acid to a nucleic acid binder
molecule (e.g., antibody, antibiotic, histone, antibody, nuclease) that is
labeled
with a fluorophore. Fluorescent labels for nucleic acids include organic
fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold
particles,
and fluorescent minerals.
The fluorescent labels preferably fluoresce at unique wavelengths when
exposed to a broadband optical source, thereby providing a method for
identification of each of the subject nucleotides in a two dimensional (2D)
spatial image.
The fluorescent labels are bound to the nucleotides, which are located on
the surfaces of the fluidic channel, and unnecessary exposure of the
fluorescent
labels to the excitation source causes photobleaching, a temporal phenomenon
where excitation of the label results in a decreased fluorescence optical
output
over time. This is a problem in the prior art where the label activation
energy is
applied, and the microscope is focused by using the fluorescent labels as the
focus target, thereby exposing the labels to photobleaching energy during the
microscope focusing interval. Because the fluorescent labels are small and the
magnification large, the range of microscope image focus is short, and the
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fluorescent labels do not appear until in the narrow range of sharp focus.
During
this time interval of microscope focusing, photobleaching is occurring, which
reduces the optical energy available for imaging the fluorescent labels,
thereby
reducing the signal to noise ratio at the detector. Additionally, the
fluorescent
label optical intensity is comparatively low, increasing the difficulty of
focusing
when using the fluorescent labels as focusing targets.
It is desired to provide a microscope which provides for focusing on an
inner surface of a fluidic channel such as one where nucleotides and
associated
fluorescent labels may collect, followed by application of fluorescence
activation
io energy to image the inner surface of the fluidic channel and associated
fluorescent labels.
In addition, imaging acquisition speed is a very important factor for the
throughput of imaging-based DNA sequencers. Traditionally, imaging time has
been shortened by increasing the number of cameras used to image multiple
regions in parallel. The invention provides an optical scheme that employs a
significantly larger field of view and a sensor that significantly improves
the
image-capturing speed without the complexity of current DNA sequencers.
Previous attempts to address this issue included TDI (time delay and
integration) line scans, where the scan speed could be fast, but required
precision
timing and high precision motion. Another attempt involved multi-imaging
heads, which required multiple detection/illumination subsystems, each with
its
own focusing mechanisms.
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Objects of the Invention
A first object of the invention is a microscope having an illuminated
fiducial pattern which is positioned a fiducial lens focal length from a
fiducial
lens, the optical energy from the fiducial lens directed to a beam splitter
and to
an objective lens positioned an adjustable distance from a flow cell having
inner
surfaces, the objective lens on the optical axis of a detector lens, the
detector lens
receiving optical energy which passes through the beam splitter and focuses
the
optical energy to a detector, the microscope thereby configured to position
the
fiducial pattern onto a change in refractive index of the flow cell sufficient
to
form a partially reflective interface and provide for focusing the microscope
onto
an inner surface of the fluidic channel.
A second object of the invention is a method for imaging the inner surface
of a fluidic channel at an interface having a change in refractive index, the
method comprising forming collimated fiducial pattern optical energy and
directing the collimated fiducial pattern optical energy to an objective lens
an
adjustable distance from the flow cell, where optical energy reflected from
the
fluidic channel interface is directed to a detector lens and focused onto a
detector, the method comprising first adjusting the adjustable distance until
the
.. fiducial pattern presents as a focused image at the detector, and
subsequently
illuminating the flow cell with optical energy operative to fluoresce labels
at an
inner surface of the fluidic channel and forming an image at the detector.
A third object of the invention is a system for detecting a discontinuity in
index of refraction forming a partially reflective optical interface, the
system
comprising a fiducial pattern generator forming a collimated image, the
collimated image directed to an objective lens such as through a beam
splitter,
the objective lens positioned a variable focal length from the discontinuity
in
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index of refraction forming a partially reflective optical interface,
reflected
optical energy from the partially reflective interface directed through the
objective lens and to a detector lens and a detector positioned a focal length
from
the detector lens.
5 A fourth object of the invention is a method for locating a surface of a
fluidic channel, the method comprising:
directing collimated optical energy from a fiducial pattern through an
objective lens positioned an adjustable distance from a surface of
the fluidic channel;
lo directing reflected optical energy from the surface of the fluidic
channel
through the objective lens through a detector lens and to a detector
positioned a detector lens focal length from the detector lens;
adjusting the distance from the objective lens to the flow cell until a
focused image of the fiducial pattern is present in the detector.
A fifth object of the invention is a method for imaging fluorescent labels
adjacent to an inner surface of a fluidic channel, the method comprising:
directing collimated optical energy from a fiducial pattern through an
objective lens an adjustable length from the inner surface of the
fluidic channel;
directing reflected optical energy from the inner surface of the fluidic
channel through the objective lens to a detector lens and to a
detector positioned a detector lens focal length from the detector
lens;
adjusting the distance from the objective lens to the fluidic channel inner
surface until a focused image of the fiducial pattern is present in
the detector;
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illuminating the flow cell with optical energy, causing the labels to
fluoresce and provide a focused image at the detector.
A sixth object of the invention is a system and method for high-resolution
and large-field imaging.
Summary of the Invention
A microscope provides for imaging fine structures such as fluorescent
labeled nucleotides at the inner surface of a fluidic channel. In particular,
the
microscope provides for the location of an upper or lower inner surface of a
fluidic channel and subsequent measurement of structures such as fluorescent
labeled nucleotides which are adjacent to the upper or lower inner surface of
the
fluidic channel.
In one example of the invention, a fluidic channel has substantially planar
upper or lower interior surfaces in a region of desired observation. The
substantially planar interior surface is within an adjustable distance which
includes the focal distance of an objective lens when the fluidic channel is
present. A detector lens is positioned on the same axis as the objective lens,
and
a detector is positioned a detector lens focal length from the detector lens.
Optionally, an illuminated image mask with a fiducial pattern is positioned a
fiducial lens focal length from a fiducial lens and substantially
perpendicular to
the axis of the objective lens. Preferably low intensity illumination energy
from
the fiducial lens is directed to a beam splitter located between the objective
lens
and detector lens, which directs the optical energy from the fiducial lens to
the
objective lens, where it forms an image of the fiducial pattern a focal length
from
the objective lens, causing focused or unfocused optical energy to be
reflected
from the discontinuity in index of refraction at the substantially planar
inner
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surface of the fluidic channel. When the objective lens is a focal length from
the
substantially planar surface of the fluidic channel, focused reflected optical
energy from the objective lens travels to the detector lens and forms a
focused
image of the fiducial pattern on the detector, providing the ability to
precisely
locate the inner surface and perform measurements with respect to that
surface.
The objective lens has a focal length which is preferably short to provide a
minimum depth of field for measurement of adjacent structures to be measured.
The combined flow cell top layer thickness and fluidic channel depth is
constrained to be smaller than the focal length of the objective to ensure the
io ability of the microscope to focus on both the upper and lower inner
surface of
the fluidic channel.
After the fluidic channel surface is located using comparatively low
intensity light for fiducial illumination, imaging is performed of the
fluorescent
features adjacent to the fluidic channel surface using high intensity optical
energy suitable for imaging fluorescent labels associated with the
nucleotides. A
focused image of the fluorescent labels is thereby provided to the detector,
and
the low intensity fiducial illumination energy prior to the application of
fluorescent label illumination energy greatly reduces undesired
photobleaching.
In addition, the invention provides a high-resolution lens system with a
substantially larger field of view than conventional microscopes, coupled with
high resolution imaging sensors with substantially larger pixel counts (>30
megapixels).
Brief Description of the Drawings
Figure 1 is a section view 100 of a microscope according to an aspect of
the invention.
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Figure 2 is a perspective view of a flow cell of figure 1.
Figure 3 is a projection view of example fiducial masks for use with the
microscope of figure 1.
Figure 4 is a section view 400 of a microscope according to another aspect
of the invention.
Figure 5A is an example fiducial mask for focusing the microscope of
figures 1 and 4.
Figures 5B, 5C, 5D, 5E are intensity profiles as measured at a detector for
objective separation distances from a flow cell.
lo Figure 6 is a checkerboard fiducial pattern.
Figure 7 is an example flow cell construction. In one embodiment, the
lower glass plate 704 can be opaque.
Figure 8A shows a detail view of a flow cell with a plurality of partially
reflective interfaces.
Figure 8B shows an example checkerboard fiducial pattern.
Figure 8C shows an example detector image of the fiducial pattern of
figure 8B.
Figure 8D shows a detail view of a fiducial of figure 8B.
Figure 9 illustrates the use of a DMD 901 to generate a fiducial pattern.
Detailed Description of the Invention
Figure 1 shows a microscope according to an aspect of the invention.
Reference coordinates x, y, and z are shown in each drawing figure for
reference
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to other drawing figures. A fluidic channel 120 is formed in transparent
housing
122, and includes a substantially planar inner surface 116. The index of
refraction for the housing 122 is selected to be different from the index of
refraction of a fluid being transported in the fluidic channel 120 by a ratio
sufficient to form a partially reflective interface, such as one returning at
least
.06% of the incident optical energy, corresponding to a difference of index of
refraction of at least 5% greater or smaller, or a minimum difference of 1%
greater or smaller index of refraction at the partially reflective interface,
returning ¨25ppm of the incident optical energy. An example reflective
io interface is formed by the case of glass (1.5) over water (1.33), and a
larger ratio
of the two refractive indices is preferable, as the ratio is proportional to
the
reflected optical energy which is directed to the detector or sensor 102 for
image
formation and the change in index of refraction forms a reflective interface
at the
glass/liquid interface. Where the inner fluidic channel interface is
encountered
in the plurality of partially reflective surfaces, each partially reflective
surface is
reflecting a percentage of the incoming optical energy according to the well-
known Fresnel ratio R = 1(n1 ¨ n2) I (n1 + n2)12
where:
n1 and n2 are the index of refraction sequence as encountered by
the incoming optical energy;
R is the coefficient of reflection returned by the partially reflective
interface. For reflective interfaces such as the fluidic channel upper
surface, the
optical energy transmitted through the subsequent optical interface T is 1¨R
for
the subsequent optical interface.
The increased proportion of reflected optical energy improves resolution
and reduces the required optical energy to perform the initial focusing of the
microscope on the fluidic channel inner surface. Additionally, the optical
energy
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of the fiducial optical source may be on the order of 1/10, 1/100, 1/1000,
1/10,000 or 1/100,000 of the optical energy required to cause the fluorescent
labels to become visible, thereby reducing the likelihood of photobleaching
while also providing features with greater contrast for focusing the
objective.
5 The improved focus accuracy thereby provides for greater accuracy and
resolution in establishing the objective lens to reflective surface focusing,
greatly
reducing the photobleaching of the fluorescent labels, since the reduced
optical
energy of the fiducial source is well below the photobleaching threshold.
Optical source 146 generates uncollimated optical energy which
10 backlights fiducial image mask 110 projecting the image mask pattern
onto
fiducial lens 108. Image mask 110 comprises patterns formed in optically
opaque and transparent features, the fiducial image mask 110 being a focal
length L2 142 from fiducial lens 108, resulting in collimated optical energy
which reflects from beam splitter 106 to objective lens 112 on axis 150, where
it
is focused at an image plane a focal length below objective 112 and reflected
by
the index of refraction discontinuity at the inner surface 116 of the fluidic
channel 120.
The fiducial image is projected into the inner surface 116, and when the
distance L3 144 from the objective lens 112 to the inner surface 116 is equal
to
the focal length of objective lens 112, a sharp image will be reflected by the
inner surface 116. When the separation distance L3 is slightly greater than
the
focal length of the objective lens 112, the image focal plane at 114 results
in the
reflection of an out-of-focus image at the inner surface 116 where the
discontinuity in refractive index (and reflective surface) is located.
Similarly, a
shorter distance L3 144 will result in a sharp focal plane at 118, whereas
optical
energy reflected from the index of refraction discontinuity at surface 116
will
similarly be out-of-focus. The particular nature of the out-of-focus fiducial
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image patterns which are reflected to the detector 102 are governed by the
well-
known circle of confusion and point spread function, and are dependent on the
particular fiducial image pattern in use.
When objective lens 112 is focused on the fiducial image in focus at inner
surface 116, reflected optical energy is collimated by objective lens 112, and
travels on optical axis 150 through beam splitter 106 to detector lens 104
(such
as a tube lens) which is a fixed focus separation Li 140 from detector 102,
thereby forming a focused image from inner surface 116 onto detector 102.
In an example embodiment, objective lens 112 focal length is variable,
io such as by moving a stage holding the flow cell assembly 120/122 with
respect
to the objective lens 112 along the z-axis shown in figure 1. Fiducial lens
108 is
a fixed focal length L2 142 from the fiducial pattern of fiducial mask 110,
and
the detector 102 is a fixed focal length Li 140 from detector lens 102.
According to this example embodiment, the displacement of the inner surface
116 such as by movement of flow cell assembly 120/122 in the z axis until a
sharp focus of the fiducial pattern occurs at detector 102 provides for a
precise
determination of the inner surface 116.
Figure 4 shows an example of the invention providing the focusing
function described in figure 1, with additional capability for multiple
wavelength
fluorescent label imaging. Reference numbers performing the same function as
the structures of other figures use the same reference numbers. The operation
of
focusing on an inner surface 116 of the fluidic channel 120 occurs as was
previously described by adjusting distance L3 144 until a sharp image of the
fiducial pattern 110 is present on a detector 102 (also referred to as a
fiducial
detector where multiple detectors are present). After the focal adjustment of
distance L3 144 is completed, an external fluorescent label optical source
(not
shown) illuminates the field of the fluidic channel 120, causing the
fluorescent
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labels associated with nucleotides on an inner surface 116 of the fluidic
channel
to emit optical energy, each fluorescent label emitting optical energy in a
unique
wavelength from other fluorescent labels, resulting in a multi-colored
fluorescent
label pattern to be directed along optical axis 150 through beam splitter 106
and
to beam splitter 103. Optical energy is directed to lens 104B to fluorescent
label
detector 102B and also to lens 104A to fluorescent label detector 102A.
Although two detectors are shown, the invention may be operative using any
number of lens/beam splitter/detector optical paths, one for each range of
wavelengths emitted by a particular fluorescent label. In an example of the
.. invention, for imaging RNA or DNA with four fluorescent labels, four
fluorescent-label optical paths and associated fluorescent label detectors may
be
used, each responsive to an associated fluorescent label. Each detector path
(comprising dichroic reflector or beam splitter, detector lens, and detector)
is
typically sensitive to a range of wavelengths associated with the emitted
wavelength of a particular fluorescent label. In one example of the invention,
beam splitter 103 has a dichroic reflective coating which reflects a specified
range of wavelengths to fluorescent label detector 102B, and passes other
wavelengths to fluorescent label detector 102A with minimal transmission loss.
In another example of the invention, a cascaded series of dichroic reflectors
103
can be provided on the optical axis 150, each dichroic reflector, lens, and
detector associated with a particular fluorescent label wavelength. In another
example of the invention for simultaneous imaging of the fluorescent labels
with
a single detector, a single multi-wavelength color detector may be used which
has sufficient spatial resolution and wavelength resolution to display the
fluorescent labels in a separable form by wavelength. For example, rather than
an RGB (red, green, blue) solid state image detector, a four or five channel
detector may be used which is specific to the particular wavelengths, or the
RGB
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channels may be linearly combined to isolate the RGB image response into the
particular fluorescent wavelengths.
In one aspect of the invention, lenses 104, 108, and 112 are anti-reflective
or have achromatic coatings as previously described. In another aspect of the
invention, the optical source 146 may be a narrowband visible optical source
such as a light emitting diode (LED) to reduce chromatic aberration and
chromatic distortion of the lenses 104, 108, and 112. In another aspect of the
invention, the image mask 110 is a quartz or glass substrate with patterned
chrome forming the fiducial pattern deposited on the substrate surface facing
io fiducial lens 108 with the patterned chrome positioned at the focal
plane of lens
108. It will be appreciated that the optical paths may incorporate additional
components such mirrors, lenses, beam splitters and optical sources, so long
as
he essential features of the optical path of the invention is maintained.
Figure 2 shows an example fluidic channel formed from a material which
is transparent to the wavelength used for fiducial illumination as well as for
the
fluorescent marker wavelengths.
Figure 3 shows example fiducial patterns 302 and 304 which may be
applied to fiducial mask 110A and 110B, respectively. Fiducial pattern 302
formed of concentric circles may be useful where it is desired to correct non-
planarity of the inner surface 116 when it is undesirably tilted with respect
to the
x-y plane, as the out of focus regions will indicate direction and angle of
the tilt
for correction. Alternatively, fiducial pattern 304 formed of an array of
lines or
other patterns that have features predominantly in the x-axis or y-axis may be
used for automatic focusing using the detector response along a single line of
detector photosensors approximately perpendicular to the array of lines. In
another aspect of the invention, the fiducial patterns may include patterns
with
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particular separation distances to enable visual measurements of structures
bound to the surface 116 in the x and y directions.
In another example of the invention, an automated focus operation is
performed by a mechanical system which adjusts the separation distance L3 144
until a minimal fiducial pattern width and maximum amplitude difference is
achieved. Figure 5A shows an example fiducial focal mask pattern, and figures
5B, 5C, and 5D show the detector response as the distance L3 is varied. An out
of focus detector response (along a single line of the 2D detector) is shown
as the
plot of figure 5B. As the distance L3 is varied closer to focus, the fiducial
detector response along this single line of the detector has the spatial
detector
response shown in figures 5C and 5D, with fiducial detector response plot 510
corresponding to optimum focus. As the distance L3 is further increased beyond
which the focus of figure 5E, the fiducial detector response progresses in
sequence to plots 508, 506, and 504.
One difficulty of an automated focus algorithm is that it may attempt to
auto-focus on the fiducial pattern 502 of figure 5A with the fiducial detector
producing the output of plot 504 for a large fraction of the focal range,
which is
indeterminate for direction of flow cell movement for optical focus. An
alternative fiducial pattern is shown in figure 6 as an alternating
checkerboard
pattern comprising fine structures and coarse structures, thereby providing a
coarse focus on the structures 602 and intervening gaps 604, after which the
focus algorithm may operate on the fiducial lines of 602 as was described for
figures 5A to 5E.
The detector 102 may be a semiconductor or solid state detector array, or
alternatively an eyepiece for direct observation. In one example of the
invention, the detector 102 is a 2D array of photosensor cells with sufficient
density of photosensor cells to form a sharp image of a focused fiducial
pattern.
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In a related example of the invention, the density of photosensor cells is at
least
4 resolution linewidths of the linewidth of a fiducial pattern focused onto
the
detector. In another related example of the invention, the photosensor cell
density is such that at least four photosensors are covered by a fiducial
pattern
5 when the microscope is focused.
The beam splitter 106 may be a dichroic coating or partially reflective
surface on an optically transmissive non-dispersive substrate such as glass.
In
one example of the invention, the reflective coating may be on the order of 5%
reflective and 95% transmissive, and the optical intensity of source 146 is
10 selected to form a reflected image at surface 116 with at least 6db
signal-to-noise
ratio (SNR).
The transparent housing 122 is preferably a material with a different index
of refraction from the index of refraction of the fluid being conveyed in
channel
120, and sufficiently different to form an optically reflective interface
sufficient
15 to form an image at the detector. Figure 7 shows an example fluidic
channel 708
formed by a void in adhesive 706 which separates upper and lower glass plates
702 and 704. In another embodiment, the lower glass plate 704 can be opaque or
relatively less transparent than the upper glass plate 702. In this example,
for
focusing the system using the fiducial optical path, the reflectance of the
air
(n1=1.0) I glass (n2=1.5) interface using Fresnel's equations is R = 1(1 ¨
1.5)/(1 +
1.5)12 = 0.04 and accordingly T= 0.96 of the optical energy continues to the
fluidic channel glass/water interface where R = 1(1 ¨ 1.5)/(1 + 1.5)12 = 0.36%
of
the remaining optical energy is reflected, of which 96% of that energy is
returned through the glass/air interface to the optical path as usable
detector
optical energy. With respect to the optical energy available to the detector,
for a
given illumination I entering the flow cell, 0.041 is reflected at the first
air/glass
interface, and 0.96 >< 0.0036 x.961= .00331 is reflected at the upper surface
of
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the fluidic channel and returned to the detector. In summary, to the detector,
the
reflection from the air/glass interface forming an artifact is ¨10x stronger
than
the desired fluidic channel inner surface reflection. These are examples of
construction for understanding the invention and are not intended to limit the
invention to the examples provided.
A disadvantage of the checkerboard pattern of figure 6 is that that where
multiple reflective interfaces are present, blurring of the fiducial pattern
602 may
occur from the out-of-focus images from the other reflective interfaces above
and below the desired reflective interface of the fluid channel which
superimpose onto the desired fiducial image from the desired reflective
interface.
In particular, with respect to figure 8A, the previously computed result shows
¨10x more optical energy returned to the detector from air/glass interface 810
than from the glass/water reflection at interface 116 of figure 8A. To address
this, figure 8B shows another example of an alternating checkboard pattern
which reduces the influence of multiple reflective layers of the flow cell,
such as
upper reflective surface 810 which is a strong reflector in the present
example,
its reflection competing with the desired inner upper reflective interface 116
which is the focusing objective, and lower reflective interface 812 of fluidic
channel 708 with spacer 706 as previously described. Objective lens 112 may
focus the fiducial image onto a desired reflective interface 116, however
upper
reflective surface 810 and lower reflective interface 812 also contribute
reflective optical energy which is superimposed onto the desired reflective
interface 116 response. The alternating checkerboard pattern of figure 8B
comprises the fiducial patterns 802 arranged such as at regular intervals
within
large open regions 804. Figure 8D shows figure 8B with detail view 820 of each
fiducial, which may be any pattern as previously described, and shown as
horizontal lines 830 in figure 8D. Figure 8C shows the resultant image at the
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detector. The advantage of using the sparsely arranged fiducial pattern
becomes
clear when viewing the resultant detector image of figure 8C, where a focused
image has the pattern 822 representing the focused pattern 830, but also
includes
a weak (comparatively dim compared to pattern 822) circle of confusion
artifact
824 from the defocused fiducial reflecting from lower surface 812, as well as
a
very strong circle of confusion artifact 826 reflecting from top surface 810,
which is returning ¨10x more optical energy than the desired fiducial image
802
as previously computed. When the microscope is focused on the reflective
surface 116, for a point source (very small fiducial extent 802 compared to
.. reflective surface separation distances), the approximate diameter of each
artifact
824 and 826 may be determined by ray-tracing geometry from lens 112 of figure
8A, such that the upper reflective surface artifact 826 may be approximated by
the intersection of rays 811 with the upper surface 810, and lower reflective
surface artifact 824 may be approximated by the intersection of rays 811 with
the
.. lower surface 812, each respectively forming a circle of confusion artifact
and
the detector, in the approximation where the fiducial extent 802 is negligible
dimension compared to the separation distance from reflective surfaces 116 to
812 or from reflective surfaces 116 to 810. The resulting circles of confusion
824 and 826 will change diameter in opposite direction while the focal point
is
changed between surfaces 810 and 812, and the dimensions of each circle of
confusion will indicate the separation distance to a desired reflective
interface
such as 116 and may be used for initial focusing. The desired reflective
interface
116 may therefore be determined from the diameter of the circle of confusion
artifacts 824 and 826 in combination with the reflective surface spacings of
the
flow cell, and thereafter the focus algorithm can change to one of finely
adjusted
using the pattern of the fiducial itself, such as 830, as was previously
described
for figures 5A to 5E. To minimize the influence of comparatively strong
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artifacts 826 on comparatively weak fiducial image 802, it may be desirable to
arrange the spacing between fiducial patterns 802 of figure 8B to ensure that
the
circle of confusion artifact 826 does not enter into an adjacent fiducial
pattern for
reasonable fluidic channel/objective separation distances. It may also be
desirable to arrange the separation distances between 810/116 and 116/812
fonning the plurality of reflective interfaces to minimize the influence of
the
circle of confusion artifacts 824 and 826 on the desired fiducial image 822.
In the present application, references to within an order of magnitude of a
nominal value include the range of 1/10th of the nominal value to 10 times the
nominal value, such as about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or
900%. A reference to an approximate value (and where "-" is used to indicate
an approximate value) is understood to be within the range of 1/2 of the
nominal
value to 2x the nominal value, such as about 60%, 70%, 80%, 90%, 110%,
120%, 130%, 140%, 150%, 160%, 170%, 180% or 190%. Although it is
preferred that the axis of fiducial lens 108 be approximately perpendicular to
the
axis of objective lens 112, any arbitrary angle of the beam splitter 106 may
be
selected which provides illumination of the fiducial image onto surface 116,
such as about 20 , 30 , 40 , 45 , 50 , 60 , 70 , 80 , 90 , 100 , 110 , 120 ,
130 ,
135 , 140 , 150 or 160 . The substantially planar region of the fluidic
channel is
understood to be sufficiently planar to provide a region of focus, such that
the
variation in diameter in the circle of confusion from one region to another
varies
by less than a factor of 10. Alternatively, the microscope may operate
correctly
where the substantially planar region of the fluidic channel is tilted from
the
optical axis, or non-planar, but with a restricted region of focus, which will
only
limit the extent of focused fiducial image and extent of focused fluorescent
label
detector image. In this example of a tilted or non-planar region,
substantially
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planar is understood to only refer to the region of the image which is focused
or
can be focused.
In another embodiment, the invention provides a high-resolution lens
system with a substantially larger field of view than conventional
microscopes,
coupled with high resolution imaging sensors with substantially larger pixel
counts (>30 megapixels).
The lens system can have the following or similar characteristics:
Wavelength: 500nm to 720nm is a useful range. Other useful wavelengths
io include a range between any of lOnm, 20nm, 50nm, 100nm, 200nm, 250nm,
300nm, 350nm (typically ultraviolet), 380nm, 400nm, 450nm, 500nm, 550nm,
600nm, 650nm, 700nm, 740nm (typically visible), 750nm, 800nm, 900nm, lgm,
10gm, 100tim, and lmm (typically infrared).
Magnification: 4x to 6x is a useful range. Other ranges include from any
of lx, 2x, 3x, or 4x to any of 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 18x, or
20x.
Magnification greater than 6x can be used with a larger imaging sensor.
Sensor resolution: 60 megapixel (mp) is a useful resolution. Other ranges
include between any of 20, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100,
110, 120, 130, 140, 150, 175, 200, 250, 300, 350, and 400 mp.
Numerical Aperture (NA) (object space): 0.5 is a typical NA. Other
ranges include between any of 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6,
0.65,
0.7, 0.75, 0.8, 0.85, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, and 1.53.
Resolution (object space): can be <1[1m, or better than 500 line pairs per
mm. Other ranges include between any of 300nm, 350nm, 400nm, 450nm,
500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1100nm, 1200nm, 1300nm,
1400nm, 1500nm, 1600nm, 1700nm, 1800nm, 1900nm, and 2000nm.
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Field of View (FOV) (Object space): 7.5mm x 5.6mm (9.4mm diameter)
are typical FOV areas. Other useful FOVs include at least 4, 5, 6, 7, 8, 9,
10, 12,
15, 20, 25, 30, 40, 45, 50, 60, 70, 80, 90, 100, 200, 500, 750, and 1000 mm2.
Top Solid support thickness: typically 170-250 lam
5 Top Solid support refractive index: typically 1.50-1.53
Aqueous layer thickness: typically 170-250 nn
Immersion Medium: Air/Dry
Tube Lens Aperture diameter: typically 35 to 60mm. Other ranges
include between any of 30, 35, 40, 45, 50, 55, 60, and 65nm. Other ranges can
io be selected to be compatible with the objective and the size of the beam
splitter.
The large field of view requires a high degree of flatness for the substrate
and thus requires more stringent manufacturing tolerances for the flow cell.
This
can be addressed by taking multiple images at different focal points and using
computational imaging algorithms to extract signal from the sample across the
15 whole field of view.
Due to the relatively low magnification and large field of view, the
fluorescent background from the thick bottom solid support and any debris
under
the flow cell can obscure detection of the signal from the sample surfaces.
This
can be significantly reduced or virtually eliminated by the use of non-
transparent
20 low-fluorescence substrate material that is also biochemically
compatible with
sequencing protocols, such as UG-1 glass (Schott AG, Mainz, Germany), which
is opaque in the visible range where sequencing imaging is carried out. The
use
of opaque glass as the solid support of the flow cell reduces the fluorescent
background.
To further discriminate signal from out-of-focal-plane background, a
patterned illumination generated by devices such as a Digital Micromirror
Device (DIN/ID) and use of computational methods.
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See, e.g., Rainer Heintzmann, "Structured Illumination Methods," pp. 265-279
in James B. Pawley (ed.) Handbook of Confocal Microscopy (Springer 2006).
The use of structured illumination further rejects out-of-focal-plane
fluorescent
background in the large field imaging based sequencing apparatus.
As shown in Figure 9, a DMD 901 can be used¨not only to generate
illumination¨but also to generate and control a fiducial pattern. Such a DMD
can be used for fiducial focusing, since multiple patterns can be configured
during imaging and focusing, which can optimize workflows by maximizing
speed, quantifying tilt, and adapting to unexpected signals.
lo The magnification and resolution of the lens system should match or
correspond to the pixel size, feature density, feature size, and the sensing
area of
the imaging sensor to optimize image acquisition speed. The illumination light
source should also produce sufficient power density and intensity uniformity
at
the sample surface(s).
As a result, the embodiment provides surprisingly low fluorescence
background and the large-field-of-view image with very high resolution.
Improved performance can be measured by total imaging time per cycle (taking
into account channel-switching and settling time), sensitivity for
distinguishing
bases, read length, and total run time. The embodiment can have applications
in
high-throughput cell imaging, such as for drug screening.
The present examples are provided for illustrative purposes only, and are
not intended to limit the invention to only the embodiments shown.
