WO2019240959A1

WO2019240959A1 - Device and method of imaging fluidic samples

Info

Publication number: WO2019240959A1
Application number: PCT/US2019/034663
Authority: WO
Inventors: Ulrich SCHAFF; Kyungjin HONG; Laura FREDRIKSEN; Jon EPPERSON
Original assignee: Sandstone Diagnostics, Inc.
Priority date: 2018-06-16
Filing date: 2019-05-30
Publication date: 2019-12-19

Abstract

A device and method for automated imaging of samples are described, particularly for non-homogeneous fluid biological samples. One purpose is particle quantification and other analysis. A device comprises a fluidic disk, a monolithic optical subsystem comprising a camera, imaging optics, rotational and focus motors, controller, and user interface. In a method, the fluidic disk receives a biological sample containing particles of interest and distributes them into multiple channels of defined thickness. Spatially distinct portions of the sample are drawn into each channel. The fluidic disk is then rotated by a motor such that a portion of each channel comes into alignment with the imaging optics and then each portion is imaged sequentially, thereby compensating for sample non-homogeneity. Embodiments include monochromatic LED illumination and image processing and particle counting in the channel images.

Description

DEVICE AND METHOD OF IMAGING FLUIDIC SAMPLES

TECHN1CAL F1ELD

[0001] This invention relates generally to imaging and analyzing fluidic samples, particularly for non-homogeneous fluid biological samples.

TECHN1CAL BACKGROUND

[0002] This invention relates to homogenization and multi-field imaging of particulate samples for particle quantification and other analysis. The maximum theoretical measurement precision for particle concentration or occurrence rate of particle sub-populations is governed by Poisson statistics lf particles are measured by direct imaging such as hemocytometiy, the

measurement precision will increase approximately with the square root of the total area imaged. However, many types of biological or other analytical samples contain an inhomogeneous distribution of particles. For example, semen is initially composed of a mixture of secretions that each has different mean sperm concentrations. Unless the sample is well homogenized, a particle concentration measurement (or measurement of a particle sub-population concentration] taken on a portion of the sample may yield results that do not reflect the bulk concentration in the entire sample. Therefore, homogenization is an important process step for several types of biological particulate analysis including semen analysis (determining concentration of sperm cells and motile sperm cells] and blood cell counts. Homogenization is typically done manually and can generate results that vary based on technique and operator training.

[0003] A typical method to reduce the detrimental effect of sample inhomogeneity is measurement and averaging of multiple replicate sub-samples. Although this method adds considerably to assay cost and labor, it helps to ensure compliance to homogenization procedures and maintains adequate measurement precision. When particle concentration is measured by direct imaging, this method may involve sequential preparation of multiple slides of known depth and sequential analysis. [0004] Particle concentration analysis by visual inspection remains a prevalent practice. Examples include hemocytometiy for sperm cells in semen and manual inspection of blood smears, e.g., for malaria. For biological samples particle concentration analysis is typically conducted with the aid of an optical microscope. Particle concentration analysis by visual inspection is highly dependent on a consistently trained operator and may have severe variance between laboratories or operators due to differences in judgment or eyesight. Visual inspection is labor intensive, leading to practical limits on the number of particles counted and visual area inspected, which limits achievable precision. Some limitations of manual microscopy may be overcome by semi-automated or fully automated systems that may ensure consistent technique, interpretation, and imaging area (e.g., number of microscope fields analyzed). Systems based on conventional microscopy are typically expensive, requiring a precision- engineered microscope, imaging system, and computer with installed operating system and analysis algorithms. Recently there have been efforts to recreate some aspects of semi-automated particle analysis systems using mobile technology. Smartphones and tablets with built-in imaging capabilities and software enable development of relatively inexpensive and portable particle analysis system. Smartphone-based diagnostic devices for testing fresh semen samples at the point-of-care have been developed for veterinary as well as home semen testing. These devices comprise a disposable microfluidic device for semen sample handling and an optical appendage containing an objective lens (a single-ball lens or two aspheric lenses depending on an optic system) and light- emitting diode (LED) for illumination. These simplified optical platforms usually offer ~ 250 magnification adequate to observe particles with a diameter of 3 to 10 pm. A few seconds-duration sperm videos are recorded and algorithmically analyzed to generate results. However, due to simplified optical design and sub- optimal components these systems suffer from inadequate optical performance and do not address the multi-field imaging and inhomogeneity adjustments required for precise analysis.

SUMMARY OF THE INVENTION [0005] A device and method for automated imaging of samples, particularly non-homogeneous fluid biological sample, such as for particle quantification and other analysis, is one embodiment. An exemplary device comprises a fluidic disk, monolithic optical subsystem comprising a camera, imaging optics, rotational and focus motors, controller, and user interface ln an exemplary method, the fluidic disk receives a biological sample containing particles of interest and distributes them into multiple channels of defined thicknesses. Spatially distinct portions of the sample are drawn into each channel. A motor then rotates the fluidic disk such that each channel comes into alignment with the imaging optics and the channels imaged sequentially, thereby compensating for sample non-homogeneity. Embodiments include

monochromatic LED illumination and image processing and particle counting in the channel images.

[0006] An embodiment includes a simplified, low-cost optical system appropriate for automated or semi-automated multi-filed microscopy. Chromatic aberrations and other weakness of single-lens objectives are overcome by the use of monochromatic LED(s) for illumination. Post-image capture processing also includes correction for coma, limited field of view, off-axis aberrations, flat field, and non-uniform illumination. Embodiments are free of multi-element objective lenses, light condenser, and adjustable optical elements ln some embodiments, the aperture is in proximity, such as 20 mm or less, to the fluidic disk.

BR1EF DESCRIPTION OF THE F1GURES

Fig 1. Disk top view showing cross-section locations A’-A’ and B-B’.

Fig 2. Disk cross sectional view A’-A’.

Fig 3. Disk cross sectional view B’-B’.

Fig 4. Disk top view during fill cross-section location C’-C’.

Fig 5. Disk cross sectional view C’-C’ during fill.

Fig 6. Optical system cross-section after fill completion.

Fig 7. Optical system top view showing cross-section location D’-D’. Fig 8. Optical system cross sectional view D’-D’.

Fig 9. Channel detail top view.

Fig 10. Channel detail top view with fiducials.

Fig 11. Channel detail top view alternate embodiment.

DETA1LED DESCRIPTION OF THE INVENTION

[0007] Descriptions, scenarios, examples and drawings are non-limiting embodiments. All references to“invention” refer to“embodiments.”

[0008] Embodiments of the invention described herein are devices intended for use in rapid automated or semi-automated quantification of particles such as bacteria, sperm, or other cells in biological samples such as semen, blood or urine. Embodiments of the device operate by the technique of hemocytometiy in which a particle count is correlated with the original concentration of particles in the biological sample. However, the device may be structured to overcome a limitation of conventional hemocytometry: limited precision due to inadequate sample homogenization.

[0009] Embodiments of the invention comprise an optical system that combines a fluidic disk with a simplified monolithic optical instrument containing all components required for multi-field particle quantification and analysis including camera, imaging optics, controllers, rotational and focus motors, a controller (such as but not limited to a single-board computer, with no restrictions on the location of the controller], and a user interface. The fluidic disk receives a biological sample containing particles of interest and distributes them into multiple channels of defined thickness. Spatially distinct portions of the sample are drawn into each channel. The disk may be rotated such that each channel comes into alignment with imaging optics and may be imaged sequentially. By averaging the particle concentration derived from each channel, the device may compensate for inhomogeneity in the sample because particle concentrations from multiple regions in the sample are assessed. Precision also improves due to the greater area imaged compared to a single image (e.g., with the square root of the number of images]. [00010] An essential aspect of embodiments of this invention is a simplified automated optical design that nonetheless achieves performance for particle analysis applications or other applications of automated multi-field microscopy. Elements of the design compensate for the limitation of low-cost components. For instance, a single-lens objective does not have corrections for chromatic aberrations. Using a monochromatic LED for illumination as discussed herein may accommodate this limitation. A single-lens objective will also have a limited field of view due to coma, flat-field, and other off-axis aberrations. The radial design allows multi-field imaging to compensate for limited field of view. The illuminator may be designed without condenser lenses by using a small aspect light source such as specific types of LEDs combined with a near-field light aperture. Finally, fixing components into place removes the necessity for adjustment elements in a conventional compound microscope. Several of these compensating design elements may be combined to achieve a system with acceptable performance.

[00011] Fig. 1 shows a top view of fluidic disk 101. The fluidic disk 101 is approximately disk-shaped and contains an inlet hole 102 and fluidly connected multiple radial channels 103 that are in fluid communication with the inlet hole 102. The inlet hole 102 may be centrally located in the fluidic disk 101. The channels 103 may have each a channel terminus 104 at their distal end and may have bonded material 105 and alignment holes 106 intervening between them. The channels 103 may be arranged radially outward from the inlet hole 102 and may be equally spaced and radially symmetric. The fluidic disk 101 may be between 25 mm and 200 mm inclusive in diameter, preferably between 60 mm and 120 mm inclusive in diameter. The inlet hole may have a size range of 5 mm to 20 mm. There may be between 2 and 20 channels and between 2 and 20 alignment holes. Fig. 1 shows the location of two cross-sections: A’- A’ and B’- B’.

[00012] Fig. 2 shows the cross section A’- A’ of the fluidic disk 101 from Fig. 1. The fluidic disk 101 may consist of a top part 201 and a bottom part 202. The top part 201 and bottom part 202 may be made from transparent plastic by injection molding or die cutting or may be made from silica-based glass or other transparent material. The inner surfaces of the top part 201 and bottom part 202 may be coated with surfactants 203 and/or hydrophilic compounds 204 to facilitate loading of fluid samples. The inner surface or the top part 201 and bottom part 202 may also be coated with enzyme 205 to facilitate modification of fluid properties such as viscosity. Enzyme 205 may be deposited on the surface within the inlet hole 102 to pre-process a fluid sample before entry into channels 103. The inner surfaces may also be coated with other types of reagents such as labeling dyes to assist sample processing or analysis. The inlet hole 102 may be cut into the top part 201 and may be the thickness of the channels 103 and the top part 201 combined. The inlet hole 102 will be thicker than the channels 103 in all cases and should have a volume capacity equal or greater than all of the channels 103 combined. The volume of the inlet chamber may be between 5 and 250 pL. The top part 201 may be 0.1 mm to 5 mm thick. The channels 103 may be 0.005mm to 0.200 mm thick. The channels 103 may preferably be 0.007 mm to 0.050 mm thick for semen analysis applications. The bottom part 202 may be 0.1 mm to 3 mm thick. The top 201 and bottom 202 parts may both be generally disk-shaped. The bottom part 202 may be 0.5 mm to 10 mm larger in diameter than the top part. The difference may also be reversed with the top part 201 0.5 mm to 10 mm larger than the bottom part 202.

[00013] Fig. 3 shows the cross section B’- B’ of the fluidic disk 101 from Fig. 1. The bonding material 105 may lie between the top part 201 and the bottom part 202. The bonding material 105 may consist of melted material from the top part 201 or bottom part 202 or may be a third component such as a sheet of pressure sensitive adhesive or thermal adhesive. The bonding material 105 may be the same thickness as the channels 103. The alignment holes 106 may extend through the full thickness of the fluidic disk 101 as show or may extend partway through such as through the bottom part 202 only.

[00014] Figs. 1 through 3 show embodiments without fluid or particles.

[00015] Fig. 4 shows a top view of a fluidic disk 101 in the process of loading a sample fluid 401 bearing particles 402. The sample fluid 401 is placed into the inlet hole 102 and is drawn from the inlet hole 102 into each of the channels 103 by capillary action and travels outward radially through the channels as shown by arrows 403. Particles 402, in the fluid 401, are drawn into the channels. Fig. 4 shows the location of cross section C’ - C’. [00016] Fig. 5 shows the cross section C’- C’ of the fluidic disk 101 from Fig. 4 in the process of fluid fill. The sample fluid 401 is drawn into channels 103 by a combination of capillary action and gravitational pressure. The capillary action may be facilitated by surfactants 203 and/or hydrophilic materials 204 coating the inner surfaces of the inlet hole, inlet hole base, inner portion of the channels, center portion of the channels, and distal portion of the channels, in any combination. Capillary action may also be facilitated by using an inherently hydrophilic material for manufacturing the fluidic disk 101 such as glass or plastic, or by hydrophilic surface treatments known in the art.

[00017] Fig. 6 shows the C’ - C’ cross section of an optical system including a fluidic disk 101 that has completed loading with sample fluid 401 and imaging assembly 601. The sample fluid 401 and suspended particles 402 travel down channels 103 until they reach the channel terminuses 104 where the fluid 401 is stopped by the discontinuation of the top part 201 due to surface tension. That is, the radius of top part 201 may be smaller than the radius of bottom part 202. Deposition of hydrophobic material on the inner surface of the bottom part 202 at the channel terminus 104 may contribute to stopping movement of fluid 401. lt should be understood that the bottom part 202 may be discontinued rather than the top part 201 as shown to achieve the fluid-stopping effect. After fluid motion ceases, the particles 402 may settle onto the inner surface of the bottom part 202, facilitating imaging in a single plane lf the particles 402 are less dense than the fluid 401, they may instead settle on the top part 201 (not shown].

[00018] The imaging assembly 601 may comprise a single, ideally aspheric, lens 602, an objective tube 603, a controller board 604 containing a camera sensor 605, and optionally a magnet 606. Embodiments may comprise a position sensor, such as a Hall-effect or optical position or rotation sensor proximal to location 606 or cam 607. The aspheric lens 602 may be a lens that is fabricated with a shape that is not a spherical section and that is a shape that focuses light of one or more wavelengths of interest (such as but not limited to visible wavelengths] without spherical aberration. Examples of such lenses are laser focus lenses such as lenses used for low cost laser pointers or laser cutters. For biological applications, the aspheric lens may have a numerical aperture of 0.10 - 0.60 and clear aperture diameter of 2 to 8 mm. Preferably, there will be no additional lenses between the aspheric lens 602 and the camera sensor 605, unlike a conventional compound objective. The objective tube 603 may be 5 mm to 200 mm in length, preferably 10 mm to 50 mm (shorter than a conventional microscope tube). The aspheric lens 602 and camera sensor 605 will preferably be centered on the same optical axis 614 (passing through light source LED 612 through the center of camera sensors 605). To reduce manufacturing cost, the controller board 604 may serve as the bottom of the enclosure for the camera sensor 605. The camera sensor may be mounted on the controller board 604.

[00019] The imaging assembly 601 may be coupled to a ferromagnetic cam 607 by way of an attached magnet 606. The magnet may be positioned below the camera board 604 or elsewhere. The imaging assembly 601 may also be coupled to the cam 607 by the attached magnet 606 and other magnets positioned on the opposite side of the cam 607. Magnetic attachment may be advantageous due to the light weight of the imaging assembly 601 compared to conventional optics. The cam 607 may be a helical cam or eccentric cam or other cam shapes for translating rotational to linear motion known in the art. The cam 607 may be made from a mixture of ferromagnetic and non-ferromagnetic material. The cam 607 may be magnetic and the attached magnet 606 replaced by ferromagnetic material. A focus motor 608 such as a stepper motor, gear motor, servomotor, or other rotational element may drive the cam 607. The focus motor 608 is ideally being a low cost stepper motor. Alternative embodiments use a piezoelectric transducer (PZT) to effect focusing, such as moving the fluidic disk 101, the focusing lens 602, the camera sensor 605, or controller board 604. ln some embodiments, the camera sensor 605 includes elements for movement for focus along the optical axis 614.

[00020] The fluidic disk 101 is connected to a rotation motor 609 by a disk interface, or disk hub 610. The rotational motor rotates the fluidic disk 101 for multi-field imaging as described later. The rotation motor 609 may preferably be the same type low cost stepper motor used as the focus motor 608.

[00021] An illuminator assembly 611 may be positioned on opposite side of the fluidic disk 101 from the imaging assembly 601. The illuminator may be located at a fixed position with respect to the fluidic disk 101, rotation motor 609 and focus motor 608. The illuminator assembly 611 may comprise an LED 612, illuminator housing 616 and aperture 613. The LED 612 and aperture 613 may be centered on the same optical axis 614 as the aspheric lens 602 and camera sensor 605. The length of the illuminator housing 616 may be 5 mm to 50 mm. LED 612 will preferably emit a narrow band of light wavelengths such as visible green (approximately 530nm) wavelengths. The LED 612 will preferably have an emitter size of less than 1 mm across to more closely represent a point light source and facilitate sharp illumination. The LED 612 will preferably have a hemispherical primary lens, a flat primary lens or no primary lens at all to project an ideal Lambertian light distribution. There are preferably no lenses between the LED 612 and the aperture 613, other than the integrated primary lens of the LED 612, and preferably no lenses between the aperture 613 and the disk 101. Alternately, a lens, preferably an aspheric lens), may be placed between the LED 612 and the aperture 613 such that the focal point of the lens matches the location of the LED 612 to parallelize the outer light rays 615. The ratio of the aperture 613 diameter to the aperture-LED distance should be less than 0.2 to facilitate mostly parallel (divergence angle less than 10 degrees) outer light rays 615 emerging from the aperture 613. The aperture 613 should be smaller in diameter than the clear aperture of the aspheric lens for best image contrast. The aperture 613 should be large enough to illuminate the microscopic field of view on the camera sensor 605. The aperture 613 is preferably located less than 20 mm from the top of the fluidic disk 101 to minimize spreading of the outer light rays 615.

[00022] The simplified optical system Fig. 6 shows illuminator assembly 611, focus motor 608, and rotation motor 609 all in fixed locations with respect to each other. This configuration provides angular and focus positioning of the fluidic disk 101, suitable for multi-field particle analysis applications without the complexity of additional components required in the prior art. Embodiments of this invention reduce cost, improve reliability, and enable use locations and less- skilled operators than prior art.

[00023] Fig. 7 shows a top view of the fluidic disk 101 coupled to the optical system. The relative position of the imaging assembly 601 and disk hub 610 are shown. The fluidic disk 101 may rest on one or more heater assemblies 701 positioned such that they do not interfere with the imaging assembly 601 or 611, or disk hub 610. lt is understood that the heater assemblies 701 may not necessarily have heating functionality and may simply provide surfaces on which to align the disk 101 during rotation. As those in the art know, there are many ways to heat, or cool, fluidic disk 101, including radiant or convection heat, controlled ambient air temperature or enclosure temperature, or use of Peltier thermoelectric heater/coolers. Resistive heaters may be used. Temperature may be controlled, possibly to body temperature Embodiments may include temperature sensors and temperature control, such as on the controller board 604. Fig. 7 shows the location of cross section D’ - D’.

[00024] Fig. 8 shows the cross section D’- D’ of the optical system. The fluidic disk 101 may connect with the disk hub 610 by one or more alignment pins 804 that pass through the alignment holes 106. Embodiments have other methods of attaching, permanently, or preferably removably, the fluidic disk 101 to the rotational drive of rotation motor 609, including but not limited to magnetic, surface tension, gravity, press-fit, electrostatic, or other mechanical or electronic alignment elements. The number of alignment pins 804 is ideally two to six. The disk hub 610 may have a surface that is parallel with the top surfaces of the heater assemblies 701 such that the fluidic disk 101 rests on both and is maintained normal to rotational axis 805 and the optical axis, shown in Fig. 6 as 614. The rotational axis 805 may pass through the center of the inlet hole 102. The fluidic disk 101 may be radially symmetric about the rotational axis 805. The axel of the rotation motor 609 and the center of the disk hub 610 may also be centered on the rotational axis 805. The heater assemblies 701 may comprise a heater board or other heater/cooler 801, a heat-conductive heat transfer layer 802 and one or more temperature sensors 803. The heater board 801 may be a printed circuit board that includes a resistive heating element. The conductive heat transfer layer 802 may comprise pressure sensitive adhesive or compound and may contain aluminum foil, silicone plastic, or other material in thermal contact with the heater 801 and with an ideal thermal conductivity greater than or equal to 0.1 W/m*K. The heat transfer layer 802 may have cutouts for electrical components, such as temperature sensors 803. The temperature sensors 803 may comprise thermistors, thermocouples, chip temperature sensors, or other temperature sensors known in the art. Heater/cooler 801 may comprise either a heater, cooler, both, or neither, in some embodiments. A heater or cooler may be located elsewhere, in some embodiments.

[00025] Fig. 9 shows a top view of two exemplary channels 103 with respect to the inlet hole 102 and rotational axis 805. The optical system may be configured to rotate the fluidic disk 101 in predetermined increments 901 (shown as 01) and 903 (shown as 03) such that the fixed optical axis 614 is aligned with sequential imaging fields 904. Rotational increment 901 is directed to sequential or proximal imaging fields within one channel 103; while channel increment 903 is directed to moving to a next channel. The fluidic disk 101 may rotate about the rotational axis 805 (normal to the plane of the Figure), presenting new regions within channels 103 to align with the optical axis 614 along channel axes 902. Each channel axis 902 runs down the center of each channel 103 , passing through the rotational axis 805. The rotational increment 901 may be a rotational angle that results in alignment of the optical axis 614 with a new imaging field 904 that does not overlap with the previous imaging field 904 within the same exemplary channel 103. Arrows 901’ show

approximately the rotation of the disk to move a new imaging field 904 into the image view region of the optical system. The imaging fields 904 may be rectangular, circular, or other shaped areas, approximately centered on the optical axis 614. The imaging field is defined by the arrangement of optical components as understood in the art and the configuration of the camera sensor ln an exemplary optical system, it is preferred to use a substantially square or circular imaging field 904 (e.g., by cropping the camera sensor data during processing) to remove optical aberrations at the periphery and reduce data processing load. The aspect ratio of the imaging field 904 will preferably be close to 1.0, such as less than 1.2: 1.0. The rotational increment 901 may be selected to maximize the number of approximately non-overlapping imaging fields 904 within a given channel 103. Anywhere between 1 and 30 inclusive imaging fields 904 may be captured for each channel 103, or a subset of all channels 103, depending on the level of measurement precision desired. The rotational increment 901 may be dynamically adjusted by the system depending on the number of particles initially detected or other factors, such as size of type of particle and type of fluid. For example, a smaller rotational increment 901 may be used if fewer particles are detected ln order to image a subsequent channel 103, the system may rotate the disk by a channel increment 903, which may be a larger angle than the rotational increment 901. ln some embodiments the rotational increment 901 may be dynamically adjustable ldeally, the channels 103 are the same length.

[00026] ln some embodiments, additional imaging fields may be used that are located radially offset from the imaging fields 904 shown in Fig. 9. Such multiple radial imaging fields may be implemented by replicating elements of the optical system, such as more than one optical axis, or by moving the fluidic disk 101 in a direction along a channel axis 902.

[00027] Fig. 10 shows a top view of a exemplary channel 103 containing fiducial marks 1001. The fiducial marks may be lines, cross hairs, spots, or other focusable features on the interior of channels 103. ln Fig. 10, a grid is shown as exemplary fiducials 1001. The fiducial marks may be applied to the bottom part 202, and may be configured to provide a target for focusing operations. The fiducial marks 1001 may be configured to be co-planar with particles 402 in a sample fluid 401 after settling of the particles 402. The fiducial marks 1001 may allow focusing operations for samples that do not contain particles 402, such as a semen sample lacking sperm, or fluid preparation failure.

[00028] Fig. 11 shows a top view of an alternative configuration and embodiment of a channel 103. The exemplary channel 103 comprises a channel entrance 1101, an imaging area 1102 with a larger width than the channel entrance 1101, and a channel exit 1103 with a smaller width than the imaging area 1102. The imaging area 1102 may be centered on the optical axis 614. This alternate configuration may increase the number of imaging fields (904 from Fig. 9] that may be captured from a given volume of sample fluid. Embodiments may contain a valve between the inlet hole 102 and the channel entrance 1101. The channel exit 1103 or the channel terminus may contain a volume for excess fluid. Alternatively, excess fluid may remain in the inlet volume. 1104 may be either a channel terminus, similar to 104, or may be a continuation of the channel, with a channel terminus being off the Figure.

[00029] Embodiments contain colored regions within channels 103 to assist in the viewing of the progress or presence of fluid through the channels 103. Additional light sources may be used to assist in such viewing.

Embodiments contain colored regions or optical filters (transmissive or reflective) aligned optically with imaging fields 904 to assist in creating, maintaining or improving the monochromaticity of light in the optical systems ln some embodiments, a lens 602, objective tube 603, or camera sensor 605 may comprise color filters, such as (but not limited to) RGB filters. Such embodiments may use image data from only sensor cells (e.g., pixels) responsive to a single color. Other such embodiments may use data from multiple sensor cells, changing the focus for each such color. For some such embodiments, narrow- band light sources may not be necessary. Embodiments include narrow-band color filters anywhere in the optical path between light source LED 612 and camera sensor 605. ln one such embodiment, such narrow-band filters, such as transmissive or reflective, may be applied to the upper part 201 or lower part 201. More than one filter may be used within the optical path.

[00030] Some embodiments comprise a lens 602, or objective tube 603, or may comprise an integral lens on, with or proximal to LED 612 with built-in focus capability. Some embodiments use multi-elements lenses. Lenses may be aspheric. Lenses may be coated.

Additional Embodiments

[00031] Alternative method of supporting and rotation the disk includes (i) use of a disk basket, in which the disk sits, wherein the disk basket is rotated by the disk motor and in turn causes the disk to rotate; and wherein the disk basket maintains the disk in a fixed disk plane.

[00032] Alternative embodiment of the disk hub: a cylindrical disk hub centered on the disk axis, driven by the disk motor; wherein the disk hub fits cooperatively in a disk recess in the bottom part. [As how a CD or DVD mounts and is driven.]

[00033] The upper part, or the lower part, or both; or a portion of the upper part or a portion of the lower part, or both, that form a surface of a channel, comprises glass. [00034] The scanning microscope comprises a housing or frame, which supports the disk motor and optical illuminator.

[00035] The scanning microscope comprises a controller.

[00036] The circuit board comprises the controller.

[00037] The scanning microscope further comprises a collar; wherein the collar maintains the objective lens in optical alignment with the optical path;

[00038] The disk inlet chamber comprises an enzyme.

[00039] The channels comprise an enzyme.

[00040] The disk inlet chamber comprises a dye.

[00041] The disk inlet chamber is adapted to support the manual addition of biologically active substance, separate from the fluid suspension. ldeal, ldeally, Optimum and Preferred— Use of the words,“ideal,”“ideally,” “optimum,”“optimum,”“should” and“preferred,” when used in the context of describing this invention, refer specifically to a best mode for one or more embodiments for one or more applications of this invention. Such best modes are non-limiting, and may not be the best mode for all embodiments, applications, or implementation technologies, as one trained in the art will appreciate.

All examples are sample embodiments ln particular, the phrase“invention” should be interpreted under all conditions to mean,“an embodiment of this invention.” Examples, scenarios, and drawings are non-limiting. The only limitations of this invention are in the claims.

May, Could, Option, Mode, Alternative and Feature— Use of the words,“may,” “could,”“option,”“optional,”“optimal,”“mode,”“alternative,”“typical,”“ideal,” and“feature,” when used in the context of describing this invention, refer specifically to various embodiments of this invention. Described benefits refer only to those embodiments that provide that benefit. All descriptions herein are non-limiting, as one trained in the art appreciates. The phrase,“configured to” also means,“adapted to.” The phrase,“a configuration,” means,“an

embodiment.” All numerical ranges in the specification are non-limiting exemplary

embodiments only. Brief descriptions of the Figures are non-limiting exemplary embodiments only.

Embodiments of this invention explicitly include all combinations and sub combinations of all features, elements and limitation of all claims.

Embodiments of this invention explicitly include all combinations and sub combinations of all features, elements, examples, embodiments, tables, values, ranges, and drawings in the specification, Figures, drawings, and all drawing sheets. Embodiments of this invention explicitly include devices and systems to implement any combination of all methods described in the claims, specification and drawings. Embodiments of the methods of invention explicitly include all combinations of dependent method claim steps, in any functional order. Embodiments of the methods of invention explicitly include, when referencing any device claim, a substitution thereof to any and all other device claims, including all combinations of elements in device claims.

Claims

DEVICE AND METHOD OF IMAGING FLUIDIC SAMPLES What is claimed is:

1. A scanning microscope comprising: a disk comprising: a top part, a bottom part, a plurality of channels located between the top part and the bottom part, and an inlet chamber fluidly connected to a first end of each channel;

a disk axis, normal to a disk surface;

a disk motor adapted to rotate the disk around the disk axis; an optical illuminator adapted to shine light into a selected channel; an imaging assembly comprising an objective lens and an image

sensor; and

an optical path passing through the optical illuminator, the disk and the imaging assembly.

2. The scanning microscope of claim 1 wherein: a second end of each channel is open.

3. The scanning microscope of claim 1 wherein: each channel is free of any chambers between the inlet chamber and the end of the each channel.

4. The scanning microscope of claim 1 wherein: each channel is radial in the disk from the inlet chamber proximal to a center of the disk to an end of each channel proximal to the perimeter of the disk.

5. The scanning microscope of claim 1 wherein: each channel comprises one or more imaging fields; wherein each imaging field passes into the optical path responsive to motion of the disk motor.

6. The scanning microscope of claim 1, further comprising: a one or more disk indexes on the disk; wherein at least one disk index identifies at least one rotational position of at least one channel of the disk.

7. The scanning microscope of claim 1 wherein: the scanning microscope is adapted such that the disk motor rotates the disk causing more than one channel, one channel at a time, to be within the optical path.

8. The scanning microscope of claim 1 further comprising: an autofocus element adapted to change the optical distance from a channel to the image sensor.

9. The scanning microscope of claim 6 wherein: the autofocus element comprises a driven cam, in contact with the imaging assembly, adapted to change the position of the imaging assembly in a translation motion along the optical path.

10. The scanning microscope of claim 1 wherein: the optical illuminator comprises no more than one aspheric single lens.

11. The scanning microscope of claim 1 wherein: the optical illuminator is free of a lens.

12 The scanning microscope of claim 1 wherein: the imaging assembly comprises no more than a single lens.

13. The scanning microscope of claim 1 wherein: an inside surface of at least channel is coated with a hydrophilic

substance.

14. The scanning microscope of claim 1 wherein: the autofocus element is adapted to focus particles attached a single surface of a channel in focus at the image sensor.

15. The scanning microscope of claim 1 further comprising: a heater and temperature sensor adapted to maintain at least one

channel at a predetermined temperature.

16. The scanning microscope of claim 1 further comprising: a disk support adapted to maintain the disk in a fixed disk plane,

wherein the disk support comprises a heater and a temperature sensor.

17. The scanning microscope of claim 1 wherein: the disk comprises an upper part and a lower part, wherein at least one portion of the upper part and lower part are in physical contact, and wherein the channels are formed by gaps between the upper part and the lower part.

18. The scanning microscope of claim 17 further wherein: a diameter of the upper part is less than the diameter of the lower part.

19. The scanning microscope of claim 1 wherein: the disk is disposable.

20. The scanning microscope of claim 1 further comprising: a circuit board;

wherein the image sensor is mounted on the circuit board;

an objective lens mount;

wherein the objective lens mount is the only mechanical support of the objective lens; and

wherein the sole mechanical support for the objective lens mount is the circuit board.

21. The scanning microscope of claim 20 further comprising: a driven cam;

wherein the driven cam is in contact with the circuit board; and wherein changing an angle of the driven cam changes the distance between the circuit board and the disk, along the optical path.

22. The scanning microscope of claim 1 wherein: the inlet chamber comprises an opening through the top part.

23. The scanning microscope of claim 1 wherein: the disk comprises an adhesive laminate layer, located between the top part and the bottom part;

wherein an upper surface of each channel is a portion of a lower surface of the top part;

wherein a lower surface of each channel is a portion of an upper surface of the bottom part; and

wherein the sides of each channel are defined by gaps in the adhesive laminate.

24 The scanning microscope of claim 1 further comprising: a disk hub connected to a shaft of the disk motor; wherein the disk hub comprises two or more hub pins;

wherein the hub pins penetrate respective holes in the disk;

wherein rotation of the disk motor causes rotation of the disk hub, around the disk axis; which then causes rotation of the disk.

25. The scanning microscope of claim 1 wherein: a fluid suspension placed into the inlet chamber flows via capillary action into all channels.

26. The scanning microscope of claim 1 wherein: the disk is adapted such that biological particles in a fluid suspension placed into the inlet chamber stick either to an upper surface of a channel or to a lower surface of the channel.

27. The scanning microscope of claim 1 wherein: at least one interior surface of the disk is coated with an enzyme.

28. The scanning microscope of claim 1 wherein: the optical illuminator is free of a lens; and

wherein the optical illuminator comprises an aperture wherein a ratio of [a diameter of the aperture] to [a distance between the aperture and an illuminating LED] is less than 0.2.

29. The scanning microscope of claim 1 wherein: each of the channels in the plurality of channels are equal length; and wherein the plurality of channels are symmetrically distributed

around the disk axis.

30. A method of biological assay comprising the steps: (a) placing a fluid suspension comprising biological particles into the inlet chamber of the device of claim 1;

(b) rotating the disk via the disk motor until a first imaging field of a first channel is in the optical path; and

(c) autofocusing via motion of the image sensor along the optical path; and

(d) capturing an image of the biological particles on an upper or lower surface of the first channel.

31. The method of biological assay of claim 30 comprising the additional steps:

(e) rotating the disk again until a second imaging field of a second channel is in the optical path; and

(f) repeating steps (c) and (d) for the second portion of the second channel.

32. The method of biological assay of claim 30 comprising the additional step:

(g) steps (b) through (d) are repeated for all imaging fields of the disk.

33. The method of biological assay of claim 30 comprising the additional steps:

(h) identifying particles within a predetermined size range; and

(i) computing a concentration of particles.

34. The method of biological assay of claim 30 comprising the additional step:

(j) contacting the biological sample with an enzyme.

35. The method of biological assay of claim 30 comprising the additional steps

(k) contacting the biological sample with a dye.

36. The method of biological assay of claim 30 comprising the additional step: (1) pulling the biological sample from the inlet into the plurality of channels by capillary action.

37. The method of biological assay of claim 30 comprising the additional step:

(1) stoppage of fluid flow into the plurality of channels after a

predetermined volume of fluid suspension has entered the channels.