阅读说明：本技术 用于定位在流体通道的内表面上的结构的显微镜 (Microscope for positioning structures on the inner surface of a fluid channel ) 是由 詹姆斯·车 车涤平 于 2020-02-05 设计创作，主要内容包括：本申请涉及用于定位在流体通道的内表面上的结构的显微镜。显微镜具有基准掩模和将准直掩模图像生成到分束器上的基准透镜,分束器将光学图像引导至物镜,在物镜处,光学图像被引导至由流体通道的内表面的折射率的变化形成的光学不连续点。反射光能通过物镜、分束器和检测器透镜被引导到检测器。当流体通道的内表面在离物镜的焦距处时,聚焦图像形成,提供在流体通道的内表面处的荧光标记物的成像。(The present application relates to a microscope for positioning a structure on an inner surface of a fluid channel. The microscope has a reference mask and a reference lens that generates a collimation mask image onto a beam splitter that directs an optical image to an objective lens where the optical image is directed to an optical discontinuity formed by a change in the refractive index of the inner surface of the fluid channel. The reflected light energy is directed to a detector through an objective lens, a beam splitter, and a detector lens. When the inner surface of the fluid channel is at a focal length from the objective lens, a focused image is formed providing imaging of the fluorescent marker at the inner surface of the fluid channel.)

1. A microscope, comprising:

a reference image mask capable of being illuminated on one surface and located at a focal length from a reference lens;

an objective lens located on a common axis with the detector lens;

a beam splitter positioned between the objective lens and the detector lens, the beam splitter capable of receiving optical energy from the reference lens and directing the optical energy to the objective lens;

a detector located at a focal length from the detector lens and capable of receiving reflected optical energy from a plurality of partially reflective surfaces, the reflected optical energy directed through the objective lens, the beam splitter, and the detector lens.

2. The microscope of claim 1, wherein the reference image mask comprises an array of lines or an array of circles.

3. The microscope of claim 2, wherein the array of lines forms an alternating checkerboard pattern.

4. The microscope of claim 1, wherein the plurality of partially reflective surfaces comprises a fluid channel having at least one flat region.

5. The microscope of claim 1, wherein at least one partially reflective surface of the plurality of partially reflective surfaces has a refractive index greater than or less than at least 1% of a different partially reflective surface.

6. The microscope of claim 1, wherein at least one partially reflective surface is formed by a fluid channel having a substantially planar inner surface.

7. The microscope of claim 1, wherein the detector is a 2D array of photodetector units operative to form a 2D image of reflected reference light energy and also of direct fluorescence marker energy from at least one of the plurality of partially reflective surfaces.

8. The microscope of claim 1, wherein the beam splitter is located at an approximately 45 degree angle relative to an axis of the objective and detector lenses.

9. A microscope, comprising:

a flow cell having a substantially planar inner surface area, the flow cell having a fluid channel operative to carry a fluid;

a reference image mask that produces a collimated beam of light and is coupled to a beam splitter that directs the collimated beam of light to an objective lens and onto a substantially flat region of the flow channel located at an adjustable distance from the objective lens;

a detector lens on a common optical axis with the objective lens and receiving reflected light energy from the substantially flat region, the reflected light energy passing through the beam splitter and then reaching a detector lens and a reference detector located at a detector lens focal length from the detector lens;

a light source for exciting a fluorescent marker in the flow cell;

one or more fluorescent marker light paths coupled to fluorescent marker light energy on the common optical axis, each fluorescent marker light path directing a particular range of wavelengths to an associated fluorescent marker detector;

the adjustable distance from the objective lens to the substantially flat region operates to provide a focused image of the fiducial pattern onto the fiducial detector;

the one or more detectors of each fluorescent marker light path operate to provide a focused image of fluorescent markers attached to the surface of the fluid channel when the light source is activated.

10. The microscope of claim 9, wherein the reference detector controls the adjustable distance to form a focused image at the reference detector.

11. The microscope of claim 9, wherein the substantially flat region of the fluid channel is an upper surface of the fluid channel.

12. The microscope of claim 9, wherein each fluorescent marker path comprises a dichroic reflector, a detector lens, and a detector.

13. The microscope of claim 9, wherein each fluorescent label light path operates to independently indicate a fluorescent label associated with at least one of adenine (a), cytosine (C), guanine (G), thymine (T), and uracil (U).

14. The microscope of claim 9, wherein the fluid path has a refractive index change of at least 10% in a region perpendicular to the substantially flat region.

15. The microscope of claim 9, wherein the reference image mask comprises an array of lines or an array of circles.

16. The microscope of claim 9, wherein the fluidic channel is bounded on at least one side by a glass surface.

17. A method for imaging a fluorescent marker in a fluid channel, the fluid channel having a reflective interface adjacent the fluid channel, the method operating in a microscope having an objective lens at an adjustable distance from the reflective interface, a fiducial pattern generator coupling a collimated fiducial pattern through the objective lens onto the reflective interface, a detector lens and a fiducial detector that receive light energy reflected from the reflective interface and form an image at the fiducial detector, and one or more fluorescent marker light paths that receive light energy from a fluorescent marker at the reflective interface, the method comprising:

activating the reference pattern generator;

adjusting a distance from an objective lens to the reflective interface until a focused image appears at the reference detector;

applying a fluorescent label light source to fluoresce the fluorescent label;

a focused image of the fluorescent marker is formed at each fluorescent marker detector in the corresponding fluorescent marker light path.

18. The method of claim 17, wherein the fiducial pattern generator is not enabled when the fluorescent marker light source is enabled.

19. The method of claim 17, wherein the fluid channel has an upper surface and the reflective interface is adjacent to the upper surface of the fluid channel.

20. The method of claim 17, wherein each fluorescent label light path is operative to independently indicate a fluorescent label associated with at least one of adenine (a), cytosine (C), guanine (G), thymine (T), and uracil (U).

21. The method of claim 17, wherein each fluorescent marker light path includes a dichroic reflector operative to reflect a particular range of wavelengths and pass other wavelengths, each dichroic reflector directing the particular range of wavelengths to a corresponding detector lens and a corresponding fluorescent marker detector.

22. The method of claim 17, wherein adjusting the distance from the objective lens to the reflective interface is performed with an alternating checkerboard reference pattern formed using a series of thin lines and large gaps, the reference detector varying the distance from the objective lens to the reflective interface until the reference detector senses a focused image based on resolving the alternating checkerboard pattern, the reference detector varying the distance from the objective lens to the reflective interface in the same direction until the lines of the checkerboard are focused.

23. The method of claim 22, wherein the reference detector uses a change in width of a reference feature and a change in peak-to-peak amplitude to determine whether to increase or decrease the distance from the objective lens to the reflective interface.

Technical Field

The present invention relates to microscopes. In particular, the invention relates to a microscope for focusing and positioning a structure at a partially reflective interface on the partially reflective interface, wherein a plurality of partially reflective interfaces are present.

Background

In certain industries (e.g., genetic sequencing and genetic research), detection of nucleotides, which are characteristic chemical moieties of nucleotides that constitute nucleic acids, is desirable. The five nucleobases adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) are referred to as primary or standard. They function as the basic unit of the genetic code, with bases A, G, C and T present in DNA and A, G, C and U present in RNA. Rare bases are also found in nature, such as 5-methylcytosine and other methylated bases, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carbonylcytosine. Other non-standard bases include isoguanine, isocytosine, and universal bases (e.g., inosine).

These nucleotides can be detected using fluorescent labeling (fluorescent labeling) specific to each type of nucleic acid base. Types of fluorescent labeling methods include direct labeling by covalent labeling of nucleic acids with fluorescent labels or by non-covalent binding or intercalation of fluorescent dyes to nucleic acids, and indirect labeling via covalent attachment of secondary labels to nucleic acids and then binding to fluorescently labeled ligand binders. Alternative indirect strategies involve binding of nucleic acids to nucleic acid binding agent molecules (e.g., antibodies, antibiotics, histones, antibodies, nucleases) labeled with fluorophores. Fluorescent markers for nucleic acids include organic fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold particles, and fluorescent minerals.

The fluorescent marker preferably fluoresces at a unique wavelength when exposed to a broadband light source, thereby providing a method for identifying each of the subject's nucleotides in a two-dimensional (2D) spatial image.

The fluorescent label binds to nucleotides located on the surface of the fluid channel, and unnecessary exposure of the fluorescent label to an excitation source causes "photobleaching", a temporal phenomenon in which excitation of the label results in reduced fluorescent light output over time. This is a problem in the prior art, where a label activation energy is applied and the microscope is focused by using a fluorescent label as a focusing target, thereby exposing the label to photo-bleaching energy during the microscope focusing interval. Because the fluorescent marker is small and the magnification is large, the range of microscope image focus is short, and the fluorescent marker does not appear until within the narrow range of sharp focus. During this time interval of microscope focusing, photobleaching occurs, which reduces the optical energy available to image the fluorescent marker, thereby reducing the signal-to-noise ratio at the detector. In addition, the fluorescent marker has a relatively low light intensity, which increases the difficulty of focusing when the fluorescent marker is used as a focusing target.

It would be desirable to provide a microscope that provides a means for focusing the interior surfaces of a fluidic channel (e.g., the surfaces on which nucleotides and associated fluorescent labels can accumulate) followed by application of fluorescence activation energy to image the interior surfaces of the fluidic channel and associated fluorescent labels.

Object of the Invention

A first object of the present invention is a microscope having an illuminated reference pattern located at a reference lens focal length from a reference lens, light energy from the reference lens being directed to a beam splitter and an objective lens located at an adjustable distance from a flow cell having an inner surface, the objective lens being on an optical axis of a detector lens that receives the light energy through the beam splitter and focuses the light energy to a detector, the microscope thereby being configured to position the reference pattern on a change in refractive index of the flow cell sufficient to form a partially reflective interface and to focus the microscope on the inner surface of the fluid channel.

A second object of the invention is a method for imaging an interior surface of a fluid channel at an interface having a change in refractive index, the method comprising forming collimated fiducial pattern light energy and directing the collimated fiducial pattern light energy to an objective lens at an adjustable distance from a flow cell, wherein light energy reflected from the fluid 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 appears as a focused image at the detector, and then illuminating the flow cell with light energy operative to fluoresce a marker at the interior surface of the fluid channel and form an image at the detector.

A third object of the invention is a system for detecting discontinuities in the refractive index forming a partially reflective optical interface, the system comprising a reference pattern generator forming a collimated image, the collimated image being directed to an objective lens, for example by a beam splitter; an objective lens located at a variable focal length from a point of discontinuity forming a refractive index of the partially reflective optical interface through which reflected light energy from the partially reflective interface is directed and reaches a detector lens and a detector located at a focal length from the detector lens.

A fourth object of the invention is a method for locating a surface of a fluid channel, the method comprising:

directing collimated light energy from the reference pattern through an objective lens located at an adjustable distance from a surface of the fluid channel;

directing reflected light energy from a surface of the fluid channel through the objective lens through the detector lens and to a detector located at a detector lens focal length from the detector lens;

the distance from the objective lens to the flow cell is adjusted until a focused image of the reference pattern appears in the detector.

A fifth object of the invention is a method for imaging a fluorescent marker adjacent to an inner surface of a fluid channel, the method comprising:

directing collimated light energy from the reference pattern through an objective lens at an adjustable length from an inner surface of the fluid channel;

directing reflected light energy from the inner surface of the fluid channel through the objective lens to the detector lens and to a detector located at a detector lens focal length from the detector lens;

adjusting a distance from the objective lens to an inner surface of the fluid channel until a focused image of the fiducial pattern appears in the detector;

the flow cell is illuminated with optical energy to fluoresce the labels and provide a focused image at the detector.

Summary of The Invention

Microscopes are used to image fine structures (e.g., fluorescently labeled nucleotides at the inner surface of a fluid channel). In particular, the microscope provides for the localization of the upper or lower inner surface of the fluid channel and the subsequent measurement of the structure (e.g., fluorescently labeled nucleotides adjacent to the upper or lower inner surface of the fluid channel).

In one example of the invention, the fluid channel has a substantially flat upper or lower inner surface in the region of desired viewing. The substantially flat inner surface is within an adjustable distance that includes a focal length of the objective lens when the fluid channel is present. The detector lens is located on the same axis as the objective lens and the detector is located at the detector lens focal length from the detector lens. The illuminated image mask with the reference pattern is located at a reference lens focal length from the reference lens and substantially perpendicular to the axis of the objective lens. Preferably, the low intensity illumination energy from the reference lens is directed to a beam splitter located between the objective lens and the detector lens, which beam splitter directs the optical energy from the reference lens to the objective lens, which forms an image of the reference pattern at the focal length from the objective lens, such that focused or unfocused optical energy is reflected due to the refractive index discontinuity at the substantially flat inner surface of the fluid channel. When the objective lens is at a focal length from the substantially flat surface of the fluid channel, focused reflected light from the objective lens can travel to the detector lens and form a focused image of the fiducial pattern on the detector, providing the ability to accurately position the inner surface and perform measurements relative to the surface. The objective lens has a preferably short focal length to provide a minimum depth of field for the measurement of the adjacent structure to be measured. The combined flow cell roof thickness and fluid channel depth is limited to less than the focal length of the objective lens to ensure the ability of the microscope to focus on the upper and lower inner surfaces of the fluid channel.

After locating the fluid channel surface using relatively low intensity light for fiducial illumination, imaging of the fluorescent feature adjacent to the fluid channel surface is performed using high intensity light energy suitable for imaging fluorescent labels associated with the nucleotides. The focused image of the fluorescent marker is thereby provided to the detector, and the low intensity reference illumination energy prior to application of the fluorescent marker illumination energy greatly reduces unwanted photo-bleaching.

Brief Description of Drawings

Fig. 1 is a cross-sectional view 100 of a microscope according to an aspect of the invention.

Fig. 2 is a perspective view of the flow cell of fig. 1.

FIG. 3 is a perspective view of an exemplary reference mask for use with the microscope of FIG. 1.

Fig. 4 is a cross-sectional view 400 of a microscope according to another aspect of the invention.

Fig. 5A is an example reference mask for focusing the microscope of fig. 1 and 4.

Fig. 5B, 5C, 5D, 5E are intensity distributions as measured at the detector for the objective lens separation distance from the flow cell.

Figure 6 is a checkerboard reference pattern.

Fig. 7 is an example flow cell structure.

FIG. 8A shows a detailed view of a flow cell with multiple partially reflective interfaces.

FIG. 8B shows an example checkerboard reference pattern.

Fig. 8C shows an example detector image of the fiducial pattern of fig. 8B.

FIG. 8D shows a detailed view of the fiducial of FIG. 8B.

Detailed description of the invention

Fig. 1 shows a microscope according to an aspect of the invention. The reference coordinates x, y, z are shown in each figure for reference to the other figures. The fluid channel 120 is formed in a transparent housing 122 and includes a substantially planar inner surface 116. The refractive index of the housing 122 is selected to be different from the refractive index of the fluid conveyed in the fluid channel 120 by a ratio sufficient to form a partially reflective interface (e.g., a partially reflective interface that returns at least 0.06% of the incident optical energy), which corresponds to a difference in refractive index at the partially reflective interface of at least greater than or less than 5%, or a minimum difference in refractive index of greater than or less than 1%, returning about 25ppm of the incident optical energy. The example reflective interface is formed by the case of glass (1.5) over water (1.33), and a larger ratio of these two refractive indices is preferred because of the ratio to be directed toThe reflected light energy used by the detector 102 for image formation is proportional and the change in refractive index forms a reflective interface at the glass/liquid interface. Where internal fluid passage interfaces are encountered in a plurality of partially reflective surfaces, each partially reflective surface is subject to the well-known Fresnel ratioReflecting a certain percentage of the incident light energy.

Wherein:

n1 and n2 are refractive index sequences as encountered by incident light energy;

r is the reflection coefficient returned by the partially reflective interface. For a reflective interface, such as the upper surface of a fluid channel, the optical energy T transmitted through a subsequent optical interface is 1-R for the subsequent optical interface.

The increased proportion of reflected light energy increases resolution and reduces the required light energy to perform initial focusing of the microscope on the interior surface of the fluid passageway. Further, the light energy of the reference light source may be on the order of 1/10, 1/100, 1/1000, 1/10,000, or 1/100,000 of the light energy required to make the fluorescent marker visible, thereby reducing the possibility of photo-bleaching while also providing features with greater contrast for focusing the objective lens. Thus, the improved focusing accuracy provides greater accuracy and resolution in establishing objective lens to reflective surface focusing, greatly reducing photobleaching of fluorescent markers because the reduced optical energy of the reference source is well below the photobleaching threshold.

Light source 146 generates uncollimated optical energy that illuminates reference image mask 110 from behind, which projects an image mask pattern onto reference lens 108. The image mask 110 includes a pattern formed in optically opaque and transparent features, and the reference image mask 110 generates collimated light energy at a focal length L2142 from the reference lens 108, which is reflected from the beam splitter 106 to the objective lens 112 on axis 150 where it is focused at an image plane at a focal length below the objective lens 112 and reflected by refractive index discontinuities at the inner surface 116 of the fluid channel 120.

The reference image is projected into the inner surface 116 and when the distance L3144 from the objective lens 112 to the inner surface 116 is equal to the focal length of the 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 reflection of the out-of-focus image at the inner surface 116 (and the reflective surface) where the discontinuity in refractive index is located. Similarly, a shorter distance L3144 will produce a clear focal plane at 118, while light energy reflected from the refractive index discontinuity at surface 116 will similarly be out of focus. The particular nature of the out-of-focus reference image pattern reflected to the detector 102 is governed by the well-known circle of confusion and the point spread function, and depends on the particular reference image pattern used.

When the objective lens 112 is focused on the reference image of the focal point at the inner surface 116, the reflected light energy is collimated by the objective lens 112 and travels on the optical axis 150 through the beam splitter 106 to the detector lens 104, the detector lens 104 at a fixed focal interval L1140 from the detector 102, thereby forming a focused image from the inner surface 116 onto the detector 102.

In an exemplary embodiment, the focal length of objective lens 112 is variable, for example, by moving a stage holding flow cell assembly 120/122 relative to objective lens 112 along the z-axis shown in fig. 1. The reference lens 108 is at a fixed focal length L2142 from the reference pattern of the reference mask 110 and the detector 102 is at a fixed focal length L1140 from the detector lens 102. According to this exemplary embodiment, accurate determination of the inner surface 116 is provided, for example, by movement of the flow cell assembly 120/122 in the z-axis until a clear focus of the fiducial pattern appears at the displacement of the inner surface 116 at the detector 102.

FIG. 4 shows an example of the present invention that provides the focusing function described in FIG. 1 with additional capability for multi-wavelength fluorescence marker imaging. The same reference numerals are used for the structures performing the same functions as those of the structures of the other drawings. The operation of focusing on the inner surface 116 of the fluid channel 120 occurs as previously described by adjusting the distance L3144 until a sharp image of the reference pattern 110 appears on the detector 102 (also referred to as a reference detector, where multiple detectors are present). After focal point adjustment of distance L3144 is complete, an external fluorescent marker light source (not shown) illuminates the field of fluid channel 120 causing the fluorescent markers associated with the nucleotides on the inner surface 116 of the fluid channel to emit light energy, each fluorescent marker emitting light energy at a unique wavelength different from the other fluorescent markers, resulting in a multi-color fluorescent marker pattern that is directed along optical axis 150 through beam splitter 106 and to beam splitter 103. The light energy is directed to lens 104B to fluorescent marker detector 102B and is also directed to lens 104A to fluorescent marker detector 102A. Although two detectors are shown, the present invention can operate using any number of lens/beam splitter/detector optical paths, one for each range of wavelengths emitted by a particular fluorescent marker. In an example of the invention, to image RNA or DNA with four fluorescent labels, four optical paths and associated fluorescent label detectors may be used, each optical path and associated fluorescent label detector being responsive to an associated fluorescent label. Each detector path (including the dichroic reflector or beam splitter, the detector lens, and the detector) is typically sensitive to the wavelength range associated with the emission wavelength of the particular fluorescent marker. In one example of the invention, beam splitter 103 has a dichroic reflective coating that reflects a particular range of wavelengths to fluorescent marker detector 102B and passes other wavelengths to fluorescent marker detector 102A with minimal transmission loss. In another example of the present invention, a cascaded series of dichroic reflectors 103 may be disposed on optical axis 150, each dichroic reflector, lens, and detector associated with a particular fluorescent marker wavelength. In another example of the present invention for simultaneously imaging fluorescent markers with a single detector, a single multi-wavelength color detector may be used with sufficient spatial and wavelength resolution to display the fluorescent markers in a wavelength-separable form. For example, instead of RGB (red, green, blue) solid-state image detectors, four-or five-channel detectors specific to a particular wavelength may be used, or the RGB channels may be linearly combined to isolate the RGB image response into a particular fluorescence wavelength.

In one aspect of the invention, lenses 104, 108 and 112 are anti-reflective or have an achromatic coating as previously described. In another aspect of the present invention, light source 146 may be a narrow band visible light source, such as a Light Emitting Diode (LED), to reduce chromatic aberration and chromatic distortion of 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 a fiducial pattern deposited on the surface of the substrate facing the fiducial lens 108, where the patterned chrome is located at the focal plane of the lens 108. It will be appreciated that the optical path may incorporate additional components such as mirrors, lenses, beam splitters and light sources as long as the basic features of the optical path of the present invention are maintained.

FIG. 2 shows an example fluid channel formed from a material transparent to the wavelength used for reference illumination and to the fluorescent marker wavelength.

FIG. 3 shows example fiducial patterns 302 and 304 that may be applied to the fiducial masks 110A and 110B, respectively. When the inner surface 116 is undesirably tilted with respect to the x-y plane, the fiducial pattern 302 formed by concentric circles may be useful in situations where correction of the non-planarity of the inner surface 116 is desired, as the out-of-focus area will indicate the direction and angle of the tilt for correction. Alternatively, the fiducial pattern 304 formed by an array of lines or other pattern having features primarily in the x-axis or y-axis may be used for automatic focusing using detector responses along a line of detector photosensors that is substantially perpendicular to the array of lines. In another aspect of the invention, the fiducial pattern may comprise a pattern having a specific spacing distance to enable visual measurement of structures bonded to the surface 116 in the x and y directions.

In another example of the present invention, the autofocus operation is performed by a mechanical system that adjusts the spacing distance L3144 until the minimum reference pattern width and the maximum amplitude difference are achieved. FIG. 5A shows an example reference focus mask pattern, and FIGS. 5B, 5C, and 5D show the detector response as the distance L3 varies. The defocus detector response (along a line of the 2D detector) is shown as the graph of fig. 5B. As the distance L3 changes closer to the focal point, the reference detector response along this line of detectors has the spatial detector response shown in fig. 5C and 5D, where the reference detector response graph 510 corresponds to the best focus. As the distance L3 increases further beyond the focus of fig. 5E, the baseline detector response progresses to graphs 508, 506, and 504 in sequence.

One difficulty with the autofocus algorithm is that it may attempt to autofocus on the reference pattern 502 of fig. 5A if the reference detector produces the output of the graph 504 for a large portion of the focus range, which is uncertain as to the direction of flow cell movement for optical focusing. An alternative fiducial pattern is shown in fig. 6 as comprising an alternating checkerboard pattern of fine and coarse structures to provide coarse focusing on the structures 602 and intermediate gaps 604, after which the focusing algorithm may operate on the fiducial lines of 602 as described for fig. 5A-5E.

The detector 102 may be a semiconductor or solid state detector array, or alternatively an eyepiece for direct viewing. In one example of the invention, the detector 102 is a 2D array of photosensors with sufficient density of photosensor cells to form a sharp image of the focus reference pattern. In a related example of the present invention, the density of the photosensor units is at least 4 resolution line widths of a line width of the reference pattern focused on the detector. In another related example of the invention, the photosensor unit density is such that at least four photosensors are covered by the reference pattern when the microscope is focused.

The beam splitter 106 may be a dichroic coating or a partially reflective surface on an optically transmissive non-dispersive substrate (e.g., glass). In one example of the invention, the reflective coating may be about 5% reflective and 95% transmissive, and the light intensity of the source 146 is selected to form a reflected image at the surface 116 having a signal-to-noise ratio (SNR) of at least 6 db.

Transparent housing 122 is preferably a material having a refractive index different from the refractive index of the fluid conveyed in channel 120 and sufficiently different to form an optically reflective interface sufficient to form an image at the detector. FIG. 7 shows an example fluid channel 708 formed by voids in the adhesive 706 that separates the upper glassA glass plate 702 and a lower glass plate 704. In this example, in order to use the reference optical path focusing system, the reflectance of the air (n1 ═ 1.0)/glass (n2 ═ 1.5) interface using the fresnel equation isAnd thus T0.96 of the light energy continues to the fluid channel glass/water interface where the residual light energy isIs reflected, with 96% of this energy being returned to the optical path through the glass/air interface as usable detector light energy. With respect to the light energy available to the detector, for a given illumination I entering the flow cell, 0.04I is reflected at the first air/glass interface, and 0.96 x 0.0036 x 0.96I-0.0033I is reflected at the upper surface of the fluid channel and returned to the detector. In summary, artifact-forming (artifact) reflections from the air/glass interface to the detector are approximately 10 times stronger than the expected fluid channel internal surface reflections. These are examples for understanding the structure of the present invention and are not intended to limit the present invention to the examples provided.

A disadvantage of the checkerboard pattern of fig. 6 is that, in the presence of multiple reflective interfaces, blurring of the reference pattern 602 may occur due to out-of-focus images from other reflective interfaces above and below the desired reflective interface of the fluid channel, which are superimposed on the desired reference image from the desired reflective interface. In particular, with respect to FIG. 8A, the results of the previous calculations show that the optical energy returning to the detector from the air/glass interface 810 is about 10 times more than the optical energy returning to the detector from the glass/water reflection at the interface 116 of FIG. 8A. To address this problem, fig. 8B shows another example of an alternating checkerboard pattern that reduces the effect of multiple reflective layers of the flow cell (e.g., an upper reflective surface 810 and a lower reflective interface 812 of the fluid channel 708 with spacers 706 as previously described), the upper reflective surface 810 being a strong reflector in this example, whose reflection competes with the desired inner upper reflective interface 116 as a focusing objective. The objective lens 112 may focus the reference image onto the desired reflective interface 116, however the upper reflective surface 810 and the lower reflective interface 812 also contribute reflected optical energy that adds to the desired reflective interface 116 response. The alternating checkerboard pattern of FIG. 8B includes fiducial patterns 802 arranged at regular intervals, for example, within a large open area 804. FIG. 8D shows a detailed view 820 of each fiducial of FIG. 8B, which can be any pattern as previously described, and is shown in FIG. 8D as horizontal line 830. Fig. 8C shows the composite image at the detector. The advantage of using a sparsely arranged fiducial pattern becomes apparent when viewing the composite detector image of fig. 8C, where the focused image has a pattern 822 representing a focused pattern 830, but also includes a weak (relatively dark compared to pattern 822) circle of confusion artifact 824 from the defocused fiducial reflected from the lower surface 812, and a very strong circle of confusion artifact 826 reflected from the top surface 810 that returns approximately 10 times more light energy than the desired fiducial image 802 as previously calculated. When the microscope is focused on the reflective surface 116, for a point source (a reference range 802 that is very small compared to the reflective surface separation distance), the approximate diameter of each artifact 824 and 826 can be determined by the ray tracing geometry from the lens 112 of fig. 8A, such that the upper reflective surface artifact 826 can be approximated by the intersection of the ray 811 with the upper surface 810 and the lower reflective surface artifact 824 can be approximated by the intersection of the ray 811 with the lower surface 812, each artifact forming in approximation a circle-of-confusion artifact and a detector, respectively, where the reference range 802 is a negligible size compared to the separation distance from the reflective surfaces 116 to 812 or from the reflective surfaces 116 to 810. As the focal point changes between surfaces 810 and 812, the resulting blur circles 824 and 826 will change diameter in opposite directions, and the size of each blur circle will indicate the separation distance to the desired reflective interface (e.g., 116) and may be used for initial focusing. Thus, the desired reflective interface 116 may be determined from the diameters of the circle of confusion artifacts 824 and 826 in combination with the reflective surface spacing of the flow cell, and thereafter the focusing algorithm may be changed to a focusing algorithm that is finely tuned using the pattern of the fiducial itself (e.g., 830), as previously described for fig. 5A-5E. To minimize the effect of the relatively strong artifact 826 on the relatively weak reference image 802, it may be desirable to arrange spacing between the reference patterns 802 of fig. 8B to ensure that the circle of confusion artifact 826 does not get into adjacent reference patterns for reasonable fluid channel/objective separation distances. It may also be desirable to provide a separation distance between 810/116 and 116/812 that forms the multiple reflective interfaces to minimize the effect of circle artifacts 824 and 826 on the desired reference image 822.

In the present application, references to values on the order of the nominal value include ranges from 1/10 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%. References to approximations (where "to" is used to indicate an approximation) are to be understood as being within the range of from 1/2 for the nominal value to 2 times the nominal value, e.g., about 60%, 70%, 80%, 90%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, or 190%. Although it is preferred that the axis of reference lens 108 be approximately perpendicular to the axis of objective lens 112, any arbitrary angle of beam splitter 106 (which provides illumination of the reference image onto surface 116) may be selected, for example, approximately 20 °, 30 °, 40 °, 45 °, 50 °, 60 °, 70 °, 80 °, 90 °, 100 °, 110 °, 120 °, 130 °, 135 °, 140 °, 150 °, or 160 °. A substantially flat region of the fluid channel is understood to be sufficiently flat to provide a focal region such that the change in diameter in the blur circle from one region to another varies by less than a factor of 10. Alternatively, the microscope may operate correctly with a substantially flat area of the fluid channel tilted from the optical axis or otherwise uneven, but with a limited focus area, which will limit only the range of the focused reference image and the range of the focused fluorescence mark detector image. In this example of a tilted or non-flat region, substantially flat is understood to refer to only the region of the image that is focused or can be focused.

The present examples are provided for illustrative purposes only and are not intended to limit the present invention to only the illustrated embodiments.