WO2024243019A1 - Methods and systems for imaging particles - Google Patents

Abstract

Provided are systems for the imaging of a particle in a system through the use of light filters to allow for the capture of transmission and scatter data simultaneously.

Description

METHODS AND SYSTEMS FOR IMAGING PARTICLES

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/503,429 filed May 19, 2023, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Flow cytometry is a commonly employed method in biological and medical research that analyzes the physical and chemical traits of cells or particles in fluid. This technique hinges on the observation of scattered light when these cells or particles cross a concentrated laser beam. The manner in which light scatters, captured at varying angles, offers key data about the analyzed particle's size, shape, and internal composition.

[0003] When a particle or cell intersects with the laser beam, the light scatters in several directions. This scattered light can be detected at forward angles (forward scatter, FSC), side angles (side scatter, SSC), and large or reverse angles (backscatter, BSC). Forward scatter is primarily informative of cell size, whereas side and backscatter provide insights into the granularity and internal complexity of the cell. Notably, forward, side and back scatter each contribute unique information about the cells or particles. In practice, collection optics like lenses or microscope objectives gather and direct the light towards optical detectors. It's crucial to mention here that these optical detectors can pick up more than one angle of light. Consequently, the light collected can be divided into two or more angles, enabling the detection of more detailed information about the cells or particles, all without increasing the number of collection optics used. This aspect is particularly critical in specific applications, such as preparing cells for sensitive purposes like cell therapy, where external labels may be undesirable.

[0004] The petite size of cells and particles, usually ranging from a few micrometers to hundreds of micrometers, necessitates exact and efficient light collection for precise flow cytometric analysis. Traditionally, microscope objectives are used to collect scattered light and guide it towards photodetectors. However, arranging multiple light-collecting components close to the sample container or fluid stream presents considerable technical and cost challenges. [0005] The compact design intrinsic to flow cytometers restricts the available space around the sample testing area, making it difficult to accommodate several objectives. Correctly positioning these elements without disrupting the fluid flow or laser path involves precision engineering, often leading to a complicated and bulky instrument design. Moreover, high-quality microscope objectives, known to enhance light collection, come at a hefty price. Including several of these elements escalates the overall cost of the flow cytometer, potentially making it less commercially viable. Ensuring perfect alignment of multiple objectives with the laser beam and sample stream can be complex and may lead to significant signal loss and deterioration of data quality if misaligned, necessitating frequent calibration and maintenance.

[0006] Taking into account these challenges, it's evident that there's a need for an approach that maximizes light collection in flow cytometry, minus the complications that come with adding more light collection elements.

SUMMARY

[0007] In some aspects provided herein, a method for imaging a particle that comprises illuminating a particle by directing an illuminating light at a region comprising the particle may be disclosed. When light encounters a particle at a 0° direction, it passes through the particle and scatters in all directions. A light collecting lens, like a microscope objective, may gather a cone of light that is scattered at small angles, or forward scattered. The extent of this scattered light depends on the numerical aperture of the lens, ranging from 0° and 30°. This light collection may be centered on the 0° angle of the illuminating light. The collected light may be further segmented according to scatter angles, which are determined by their distance from the central axis of the lens. This segmentation may occur at the back aperture of the lens (in the case of a microscope objective), facilitated by one or more optical elements such as lenses, prisms, gratings, mirrors, and masks. In a first case, the first division of the collected light may exhibit a small scatter angle ranging about from 0° to 10° and may be directed towards a first detector to produce a brightfield image. In a second case, a second division of light may be a small angle scatter of about 10° to 30° that may be directed towards a second detector to generate a darkfield image.

[0008] The incident light may then generate a signal light which may be passed through optical elements, splitting the signal light into a first optical signal and a second optical signal. The first optical signal may then be passed through a first light filter, the first light filter selectively allows a portion of the first optical signal through. The second optical signal may then be passed through a second light filter, the second light filter selectively allows a portion of the second optical signal through. The method may further separately detect both the first optical signal and the second optical signal and creating an image from the composite of the first optical signal data and the second optical signal data. [0009] In some embodiments, the first light filter and second light filter may comprise a blocking element. In some embodiments, the blocking element may be an adjustable aperture. In some embodiments, the blocking element may be a pinhole aperture. In some embodiments, the blocking element may be in a shape chosen from among a circle, an annulus, a square, a rectangle, a triangle, an oval, or a polygon.

[0010] In some embodiments, the blocking element may comprise an inner portion and an outer portion. In some embodiments, the inner portion may comprise a circular shape and the outer portion may comprise a circular shape which may define an annulus region through which an optical signal may be able to pass through.

[0011] In some embodiments, the signal light may be split using a half-metal mirror. In some embodiments, the illuminating light may comprise one or more LEDs. In some embodiments, the illuminating light may comprise one or more lasers.

[0012] In some embodiments, the illuminating light may comprise at least two lasers. In some embodiments, the at least two laser may comprise a 561 nm laser and a 488 nm laser. In some embodiments, the first optical signal may comprise a 561 nm wavelength light and the second optical signal may comprise a 488 nm wavelength light.

[0013] In some embodiments, the lens may have a Numerical Aperture of at least 0.28.

[0014] In some embodiments, the refractive lens may be an objective lens. In still another embodiment, the refractive lens may be achromatic lens, a plano-convex, or double convex lens. [0015] In some embodiments, the first optical signal and the second optical signal may be different wavelengths.

[0016] In some embodiments, the adjustable aperture size may range from 1 mm and 11 mm.

[0017] In some embodiments, the first optical signal may comprise a darkfield signal. In some embodiments, the first optical signal may comprise a forward scattering signal.

[0018] In some embodiments, the first optical signal may comprise a 0°+/- 30° forward scattering signal. In some embodiments, the first optical signal may comprise a 5° forward scattering signal.

[0019] In some embodiments, the first optical signal may comprise a back scattering signal. In some embodiments, the first optical signal may comprise a 180°+/- 30° back scattering signal. [0020] In some embodiments, the second optical signal may comprise a brightfield or transmission signal. In some embodiments, the second optical signal may comprise a scattered signal. [0021] In some embodiments, the first light filter, the second light filter, or both may be attached to a filter wheel. The filter wheel may be rotated such that the blocking element may be selected from among multiple blocking elements.

[0022] In some embodiments, the method may further comprise splitting the signal light into an nth optical signal and passing the nth optical signal through a nth light filter. The nth light filter may selectively allow a portion of the nth optical signal through. The nth optical signal may be detected and an image from the composite of the first optical signal data, the second optical signal data, and the nth optical signal data may be created. In some embodiments, the nth optical signal may be chosen from among a 0°+/- 30° forward scattering signal, 180°+/- 30° back scattering signal.

[0023] In some aspects provided herein, a system for imaging of moving particles may be provided. In some embodiments, the system may comprise a particle motion device including a substrate to allow particles to move along a travel path in a first direction.

[0024] In some embodiments, the system may comprise an optical illumination system to scan with a light in a region of the travel path of a particle. In some embodiments, the system may comprise an optical detection system optically interfaced with the particle motion device and operable to obtain optical signal data associated with the particle.

[0025] In some embodiments, the optical detection system may include one or more beam splitters, one or more photodetectors, an adjustable aperture and one or more light filters positioned between the particle motion device and the one or more photodetectors. In some embodiments, the one or more light filters may be designed to selectively allow a portion of the optical signal to pass through the light filters and may be detected by the one or more photodetectors.

[0026] In some embodiments, the system may comprise a data processing unit in communication with the optical detection system, the data processing unit may include a processor or FPGA (Field Programmable Gate Array) or a combination of one or more processors such as GPU, CPU or FPGAs configured to process the optical signal data obtained by the optical detection system and produce data including information indicative of the features of the particle.

[0027] In some embodiments, the light filters and adjustable aperture may be in parallel with each other, and the one or more light filters may selectively allow a scatter optical signal though.

[0028] In some embodiments, the optical illumination system may further comprise an LED. In some embodiments, the optical illumination system may further comprise a laser. In some embodiments, the optical illumination system may further comprise a combination of one or more laser or LED light sources. In some embodiments, the optical illumination system may scan the region of the travel path of the particle with both laser simultaneously.

[0029] In some embodiments, the adjustable aperture may remove the scatter optical signal. In some embodiments, one or more of the light filters may comprise a blocking element in a shape chosen from among a circle, a square, a rectangle, a triangle, an oval, or a polygon. In some embodiments, the one or more light filters may comprise a blocking element comprises an inner portion and an outer portion.

[0030] In some embodiments, the inner portion may comprise a circular shape and the outer portion may comprise a circular shape defining an annulus region through which the optical signal may be able to pass through.

[0031] In some embodiments, at least one of the one or more light filters may selectively allow a 0°+/- 30° forward scatter portion of the optical signal through. In some embodiments, at least one of the one or more light filters may selectively allow a 5° forward scatter portion of the optical signal through. In some embodiments, at least one of the light filters may selectively allow a 30° forward scatter portion of the optical signal through. In some embodiments, at least one of the light filters may selectively allow a 180°+/- 30° back scatter portion of the optical signal through. In some embodiments, at least one of the one or more light filters may selectively allow a 5° back scatter portion of the optical signal through. In some embodiments, at least one of the one or more light filters may selectively allow a 30° back scatter portion of the optical signal through. In some embodiments, the particle may lack a fluorescent label.

[0032] In some aspects provided herein, an image-based particle sorting system may be disclosed. In some embodiments the image-based particle sorting system may comprise a particle flow device structured to include a substrate, a channel formed on the substrate operable to flow particles along a flow direction to a first region of the channel, and one or more output paths branching from the channel at a second region proximate to the first region in the channel. [0033] In some embodiments the image-based particle sorting system may comprise an imaging system interfaced with the particle flow device and operable to obtain image data associated with a particle when the particle may be the first region during flow through the channel. In some embodiments, the imaging system may comprise one or more light filters that selectively filter the image data and simultaneously generate transmission image data and one or more scatter image data. In some embodiments the image-based particle sorting system may comprise a data processing and control unit in communication with the imaging system. [0034] In some embodiments, the data processing and control unit may include a processor configured to process the transmission image data and one or more scatter image data obtained by the imaging system to determine one or more properties associated with the particle from the processed image data and to produce a control command based on a comparison of the determined one or more properties with a sorting criteria. In some embodiments, the control command may be produced during the particle flowing in the channel and may be indicative of a sorting decision determined based on one or more interparticle spatial attributes ascertained from the image signal data that corresponds to the particle. In some embodiments the particle may lack a fluorescent label.

[0035] In some embodiments the image-based particle sorting system may comprise an actuator operatively coupled to the particle flow device and in communication with the actuator. In some embodiments, the actuator may be operable to direct the particle into an output path of the two or more output paths based on to the control command.

[0036] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0037] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0039] FIG. 1 depicts a diagram using an entry and exit lens to scan a particle with a beam of light and separate the signal light beam into two portions, direct transmission and scatter.

[0040] FIG. 2A depicts an exemplar light filter that blocks the direct transmission portion of the signal light beam. FIG. 2B depicts an exemplar light filter that blocks the scatter portion of the signal light beam.

[0041] FIG. 3 depicts a diagram of a system that uses different light filters to collect direct transmission data and scatter data simultaneously.

[0042] FIG. 4 depicts a diagram of a system that uses different light filters to collect direct transmission data and scatter data simultaneously.

[0043] FIG. 5A depicts a 488 nm brightfield (transmission) aperture alignment and test using a 2 mm diameter aperture and HEK cells. FIG. 5B depicts a 488 nm brightfield (transmission) aperture alignment and test using a 4 mm diameter aperture and HEK cells. FIG. 5C depicts a 488 nm brightfield (transmission) aperture alignment and test using a 5.5 mm diameter aperture and HEK cells. FIG. 5D depicts a 488 nm brightfield (transmission) aperture alignment and test using a 6.5 mm diameter aperture and HEK cells. FIG. 5E depicts a 488 nm brightfield (transmission) aperture alignment and test using a 7.5 mm diameter aperture and HEK cells. FIG. 5F depicts a 488 nm brightfield (transmission) aperture alignment and test using a 10 mm diameter aperture and HEK cells.

[0044] FIG. 6A depicts a 488 nm brightfield (transmission) aperture alignment and test using a 2 mm diameter aperture and rainbow beads. FIG. 6B depicts a 488 nm brightfield (transmission) aperture alignment and test using a 5.5 mm diameter aperture and rainbow beads. FIG. 6C depicts a 488 nm brightfield (transmission) aperture alignment and test using a 10 mm diameter aperture and rainbow beads.

[0045] FIG. 7A depicts an exemplar light filter in a rectangular confirmation for blocking the transmitted light and allowing the scattered light through. FIG. 7B depicts another exemplar light filter in a rectangular confirmation for blocking the transmitted light and allowing the scattered light.

[0046] FIG. 8A depicts a 488 nm brightfield (transmission) rainbow bead test. FIG. 8B depicts a 561 nm darkfield (transmission) rainbow bead test using the light filter of FIG. 7A.

[0047] FIG. 9A depicts a 488 nm brightfield (transmission) rainbow bead test. FIG. 9B depicts a 561 nm darkfield (transmission) rainbow bead test using the light filter of FIG. 7B. [0048] FIG. 10A depicts a 488 nm brightfield (transmission) HEK cell test. FIG. 10B depicts a 561 nm darkfield (transmission) HEK cell test using the light filter of FIG. 7A.

[0049] FIG. 11A depicts a 488 nm brightfield (transmission) HEK cell test. FIG. 11B depicts a 561 nm darkfield (transmission) HEK cell test using the light filter of FIG. 7B.

[0050] FIG. 12 depicts multiple possible annulus shaped light filters.

[0051] FIG. 13A and FIG. 13B depict tests using an annulus shaped light filter for imaging of a 10 pm rainbow bead using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image.

[0052] FIG. 14 depicts the correlation between the transmission images and the forward scatter images of FIG. 13A and FIG. 13B.

[0053] FIG. 15A and FIG. 15B depict tests using an annulus shaped light filter for imaging of a HEK cell with a 1 pm envy green bead using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image.

[0054] FIG. 16 depicts the correlation between the transmission images and the forward scatter images of FIG. 15A and FIG. 15B.

[0055] FIG. 17A and FIG. 17B depict tests using an annulus shaped light filter for imaging of a wild type HEK293 cell using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image.

[0056] FIG. 18 depicts the correlation between the transmission images and the forward scatter images of FIG. 17A and FIG. 17B.

[0057] FIG. 19A and FIG. 19B depict tests using an annulus shaped light filter for imaging of a GFP HEK293 cell using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image.

[0058] FIG. 20 depicts the correlation between the transmission images and the forward scatter images of FIG. 19A and FIG. 19B.

[0059] FIG. 21A and FIG. 21B depict tests using an annulus shaped light filter for imaging of a Veri-Cell human leukocyte™ using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image.

[0060] FIG. 22 depicts the correlation between the transmission images and the forward scatter images of FIG. 17A and FIG. 17B.

[0061] FIG. 23A and FIG. 23B depict tests using an annulus shaped light filter for imaging of a GFP algae using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image. [0062] FIG. 24 depicts the correlation between the transmission images and the forward scatter images of FIG. 23A and FIG. 23B.

[0063] FIG. 25A and FIG. 25B depict tests using an annulus shaped light filter for imaging of a GFP algae treated with an antibody using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image.

[0064] FIG. 26 depicts the correlation between the transmission images and the forward scatter images of FIG. 25A and FIG. 25B.

[0065] FIG. 27A and FIG. 27B depict tests using an annulus shaped light filter for imaging of a wild type algae using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image.

[0066] FIG. 28 depicts the correlation between the transmission images and the forward scatter images of FIG. 27A and FIG. 27B.

[0067] FIG. 29A depicts tests using a different sized annulus shaped light filter for imaging of a rainbow beads using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image. FIG. 29B depicts an exemplar signal over time graph that shows 561 scattering signal as the bead was flowed through the flow cell taken from one of the tests in FIG. 29A.

[0068] FIG. 30A depicts tests using a different sized annulus shaped light filter for imaging of a rainbow beads using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image. FIG. 30B depicts an exemplar signal over time graph that shows 561 scattering signal as the bead was flowed through the flow cell taken from one of the tests in FIG. 30A.

[0069] FIG. 31 A depicts tests using a different sized annulus shaped light filter for imaging of a rainbow beads using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image. FIG. 31B depicts an exemplar signal over time graph that shows 561 scattering signal as the bead was flowed through the flow cell taken from one of the tests in FIG. 31A.

[0070] FIG. 32 A depicts tests using a different sized annulus shaped light filter for imaging of a rainbow beads using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image. FIG. 32B depicts an exemplar signal over time graph that shows 561 scattering signal as the bead was flowed through the flow cell taken from one of the tests in FIG. 32A.

[0071] FIG. 33A depicts tests using a different sized annulus shaped light filter for imaging of a rainbow beads using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image. FIG. 33B depicts an exemplar signal over time graph that shows 561 scattering signal as the bead was flowed through the flow cell taken from one of the tests in FIG. 33A.

[0072] FIG. 34A depicts tests using a different sized annulus shaped light filter for imaging of a rainbow beads using a 488 nm laser for a direct transmission image and a 561 nm laser for the forward scatter image. FIG. 34B depicts an exemplar signal over time graph that shows 561 scattering signal as the bead was flowed through the flow cell taken from one of the tests in FIG. 34A.

[0073] FIG. 35A and FIG. 35B depict diagrams of systems using a dark filed mask and a bright field mask.

[0074] FIG. 36 depicts the selection of location of dark field and bright field masks depending on the transmitted beam intensity profile.

[0075] FIG. 37 depicts an example of a forward scattering light blocking disk design.

[0076] FIG. 38A depicts a diagram of a system using an annulus mirror.

[0077] FIG. 38B depicts an example of an annulus mirror design.

[0078] FIG. 39A and 39B depict an image of an example annulus mirror.

DETAILED DESCRIPTION

[0079] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

DEFINITIONS

[0080] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0081] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0082] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.

[0083] The terms "brightfield image" or "transmission image" are used to describe images where the source of illumination produces a bright backdrop. In instances where scattered light is utilized to generate images, it usually occurs at a small angle relative to the main axis of illumination. This positioning allows the light from the source of illumination to reach the detector directly.

[0084] The term "darkfield image," as used in this disclosure, denotes an image where the illumination source doesn't generate a bright backdrop. When employing scattered light to create images, it typically happens at a larger angle compared to the main axis of illumination. In such cases, the light from the illumination source may not be able to reach the detector directly. Instead, predominantly the light that is scattered from a cell or particle has the ability to reach the detector.

[0085] As used herein, the term "optical element" refers to any component that modifies the path or quality of light. It includes, but is not limited to, a lens, multiple lenses, an objective lens, a mirror, a prism, a mask, a grating, a polarizer, a collimator, an aperture, a filter, and any other element that serves a similar function.

[0086] The term “light filter” refers to a device or material designed to selectively transmit, absorb, or reflect light of specific wavelengths or wavelength ranges at distinct locations along the optical path. The purpose of a light filter is to modify both the spectral properties and the spatial propagation of light. It accomplishes this by allowing light of certain wavelengths at specific locations to pass through while obstructing others.

[0087] The terms "light scatter" or "scatter" refer to the phenomenon observed when a cell or particle is illuminated by a laser or other light source. Some of this light may pass through the cell or particle with little to no interaction, resulting in what is termed as 0° of scatter. Furthermore, light can be scattered in all directions, illustrating the wide-ranging interactions it can have with the illuminated particles or cells.

[0088] As used herein, the terms "small angle scatter" and "forward scatter" refer to the scattering of light within a range of 0° ± 90°. Specifically, a cone of forward scattered light reaches the aperture of a first microscope objective. The range of light angles acceptable by the microscope objective is determined by its Numerical Aperture (NA). Light within the forward direction of 0° ± 30° can be collected. Theoretically, light collection could be broadened to a range of 0° ± 90°. The term "side scatter" refers to light scattered approximately at a 90° angle. The terms “large angle scatter” or “backscatter” are used to describe light scatter within the span of 180° ± 90°. In this instance, a cone of backscattered light reaches the aperture of a second microscope objective. In this disclosure, light in the backward 180° ± 30° direction may be collected. In theory, light could be gathered from a wider range of 180° ± 90°. Images from this process can be classified as a darkfield image, backscatter image, large angle scatter image, backscatter darkfield image, or large angle scatter darkfield image. It is feasible that for the purposes of this disclosure, the backscattered light can be divided into separate sections to generate additional information or images.

EXAMPLE OPTICAL SYSTEMS

[0089] Imaging devices often are utilized to detect and optionally sort particles according to light emitted by the particles and/or light that has interacted with the particles (e.g., light diffracted, scattered and/or reflected by particles). Light is electromagnetic radiation of any wavelength or frequency. The value for the wavelength or frequency generally is for light propagating through a vacuum. Light can be characterized as visible light, ultraviolet light and/or infrared light in some embodiments. Visible light generally is of a wavelength of about 390 nanometers to about 750 nanometers, and generally is of a frequency of about 400 terahertz (THz) to about 790 THz. Infrared light generally is of a wavelength of about 0.74 micrometers to about 300 micrometers, and generally is of a frequency of about 300 gigahertz (GHz) to about 400 THz (near infrared often is about 120 THz to about 400 THz; mid infrared often is about 30 THz to about 400 THz; and far infrared often is about 300 GHz to about 30 THz). Ultraviolet light generally is of a wavelength of about 10 nanometers to about 400 nanometers, and generally is of a frequency of about 0.75 petahertz (PHz) to about 30 PHz (near ultraviolet often is about 400 nm to about 300 nm, mid ultraviolet often is about 300 nm to about 200 nm, and far ultraviolet often is about 200 nm to about 122 nm). A photon is a quantum of light, and a photon can have a particular photon energy.

[0090] In some embodiments, a particle may be an agent that emits light (e.g., a fluorophore) In some embodiments, a particle may be a complex of molecules that comprises an agent that emits light. In some embodiments, a particle includes but is not limited to one or more biological agents (e.g., cell, protein, nucleic acid, biological membrane (e.g., vesicle, liposome, the like and combinations thereof)). A particle can comprise in some embodiments one or more antibodies in association with one or more biological agents (e.g., bound to a biological agent). An antibody sometimes may be linked to an agent that emits light. A combination of different particles can be present in a device. A combination of different particles sometimes comprises different particles that emit different wavelengths of light.

[0091] In some embodiments, light introduced by a light source may be transmitted through a wall, aperture, lens, slide or combination thereof into the illumination region. The angle of light emitted by a light source can be at an angle with respect to the illumination region suitable for illuminating a particle within the device. In certain embodiments, a particle can interact with light introduced into the illumination region, and light that has interacted with the particle and may be scattered, reflected or diffracted by the particle can be transmitted from the illumination region to one or more other components in the imaging device.

[0092] In some embodiments, a particle in an illumination region can emit light of a particular wavelength or in a particular wavelength range, and all or a portion of the wavelength range can be transmitted from the channel to one or more other components in a flow cytometry device. A particle sometimes emits light of a particular wavelength or wavelength range, which wavelength or wavelength range can be different than the wavelength or wavelength range emitted by a light source (e.g., excitation wavelength(s) emitted by the light source may excite a fluorophore particle or fluorophore attached to a particle and the fluorophore may emit light of different wavelength(s)). Light emitted by a particle, or that has interacted with a particle, can transmit through a or be conducted by one or more intermediary structures, to a detector. Nonlimiting examples of intermediary structures include a mask, light filter , waveguide, mirror, lens, dichroic filter, prism, photo diffractive component (e.g., diffraction grating), the like and combinations thereof.

[0093] An imaging device may include an optical filter, a reflector or combination thereof. A device may include one or more optical filters, non-limiting examples of which include absorptive filter, dichroic filter, monochromatic filter, infrared filter, ultraviolet filter, neutral density filter, longpass filter, bandpass filter, shortpass filter, guided-mode resonance filter, metal mesh filter, polarizer filter, optical notch filter (e.g., precision optical notch filter) the like and combinations thereof. A device may include one or more components that reflect light, nonlimiting examples of which include flat mirrors, curved surface mirrors, parabolic surface mirrors, partial metal mirrors, and dichroic mirrors. A mirror sometimes substantially reflects light of a particular wavelength range and may be substantially transparent to, and does not reflect, light of a different wavelength range. A mirror in some embodiments, substantially reflects light in a wavelength range that excites a fluorophore (e.g., a fluorophore particle or fluorophore linked to or associated with a particle) and may be substantially transparent to light in a wavelength range emitted by the excited fluorophore.

[0094] Light emitted from, or light that has interacted with, a particle in a illumination region sometimes may be transmitted from a illumination region to a light filter. An light filter often includes two or more zones (e.g., about 2, 3, 4, 5, 6, 7, or more zones). An light filter sometimes includes a mask comprising substantially transparent zones and substantially opaque zones (e.g., optical apertures, bands).

[0095] An light filter sometimes may be located a certain distance from a illumination region in which a particle may be present, and sometimes a distance of about 1 centimeter (cm) to about 100 cm (e.g., about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90 cm). Light emitted by a channel sometimes may be transmitted through one or more other components (e.g., lens, mirror) before the light, or modified version thereof, contacts a light filter. Zones sometimes may be discrete zone segments on a filter. A non-limiting example of a light filter that includes discrete zones is illustrated in FIG. 12.

[0096] A light filter often includes one or more zones that transmit substantially all of the light that may be transmitted to the filter (e.g., all pass filter zone). A light filter generally includes two or more zones that transmit a portion of the light transmitted to the filter, where at least one zone transmits a different portion of light than another zone. An light filter sometimes performs as broad pass, continuous, band pass filters, the like or combinations thereof. A different portion of light sometimes may be a different wavelength subrange of a different energy subrange of, and/or a different frequency subrange of, the light wavelength range, light energy range and/or light frequency range, respectively, received by the light filter. An light filter sometimes includes one or more zones that transmit substantially none of the light transmitted to the light filter (e.g., substantially opaque zone). An light filter comprising multiple zones sometimes is referred to as a filter with a “series of optical filters” and an “array of optical filters” herein, where each of the optical filters in the series or the array may be a zone. [0097] An light filter and zones of a zones of an light filter can have dimensions suitable for detecting a particle, determining velocity of a particle, determining size of a particle and/or detecting wavelength(s) of light emitted or that have interacted with a particle, for example. In some embodiments, an light filter may be substantially circular and includes suitably shaped zones distributed around the circular structure for transmitting light (e.g., circular, ovoid, rectangular, square, triangular, segment of the circle).

[0098] In some embodiments, an light filter may be substantially rectangular and includes substantially two zones across the longer rectangular dimension that permit the transmission of light. The light filter may define two slits on the top and bottom of the rectangle that permit the transmission of light. In such embodiments, the rectangular light filter would comprise a height and a width within the dimensions of the collection lens. The light filter would further define a diameter for the aperture thus defining a region that would allow passage of light through the aperture.

[0099] In some embodiments, an light filter may be substantially circular and includes substantially two zones, an inner circle that blocks the transmission of light and an outer circle that permit the transmission of light. In such embodiments, the circular light filter would comprise an inner diameter and an outer diameter within the dimensions of the objective lens. The light filter would further define a diameter for the aperture thus defining a region that would allow passage of light through the aperture.

[0100] In some embodiments, an light filter may be substantially circular and includes substantially three zones, an inner circle that blocks the transmission of light and an outer circle that permit the transmission of light and a further outer circle that blocks the transmission of light. In such embodiments, the light would be permitted to pass through a filter zone in the shape of an annulus. The light filter would further define a vertical radius, a horizontal radius, and an area for the inner zone oval. Thus, when combined with the same parameters defined for the outer zone, would define a definite shape and size for the annulus shaped transmission zone. [0101] In some embodiments, the light filter may be the inverse of the light filter for any of the previously disclosed light filters. In such an embodiment, any zone that was defined as allowing the transmission of light would block the transmission of light and vice versa.

[0102] A light filter can be manufactured by any suitable process known in the art. Zones with different transmission properties may include different agents or one agent in different amounts, for example.

[0103] A light filter may comprise multiple layers. A light filter sometimes includes a support structure on which one or more coating layers may be deposited. Any suitable structure or support structure can be utilized, and non-limiting examples include glass, polymers and the like. Each zone independently may be of substantially uniform thickness or varying thickness (e.g., stepped thickness, tapered or flared thickness (e.g., substantially uniform taper or flare). Each zone independently may include one or more coatings (e.g., same or different materials in each coating) and/or one or more layers (e.g., same or different materials in each layer). A zone comprising multiple layers may include alternating layers, each layer comprising different materials. Each coating or layer in a zone may have the same refractive index or may have different refractive indices. Zones of a light filter that transmit different wavelength ranges of light may have the same refractive index or may have different refractive indices. Zones that transmit different wavelengths of light sometimes have a different number of layers, different materials, different thicknesses, the like or combination thereof. Where adjacent zones have different thicknesses, the transition from one thickness to another may be any suitable transition, such as stepped, tapered or flared for example.

[0104] In some embodiments, an imaging device comprises a splitter that effectively receives light emitted by, or light that has interacted with, a particle in an illumination region. The light emitted from the illumination region may be transmitted through one or more other components in the device (e.g., lens, filter) prior to the splitter receiving such light. A splitter can split received light into two or more split beams. Each of the two or more split beams sometimes may be directed to a separate light filter. Thus, an imaging device sometimes includes two or more light filters, and each or the light filters sometimes includes zones that transmit different amounts of light than zones in other light filters. Light in one split beam can be of the same wavelength range or different wavelength range as light in another split beam. Non-limiting examples of splitters include those that comprise two triangular glass prisms, half-silvered mirrors and dichroic mirrored prisms.

[0105] A light filter in an imaging device sometimes includes no mirrored surfaces, and in some embodiments, a light filter may be not a Fabry -Perot cavity filter or Fabry -Perot etalon. In certain embodiments, a light filter in a cytometry device may be not a Bragg reflector, which Bragg reflector is defined as having multiple layers and reflects light having a wavelength about four times the optical thickness of the layers.

[0106] A light filter often may be not directly in contact with a photodetector component of a device. A light filter often may be located a certain distance from a photodetector component surface, for example a distance of about 0.1 cm to about 20 cm away from a photodetector component surface (e.g., about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 cm). In certain embodiments, one light filter may be oriented with one photodetector such that light transmitted by the light filter may be transmitted to the one photodetector and no other photodetector. A device includes no photodetector array in some embodiments, and one light filter may be in detectable association with one photodetector and no other sensor cells of a photodetector array. A photodetector component surface often may be not directly in contact with, and often not distributed along, an illumination region in an imaging device, and often may be located a certain distance from the illumination region.

[0107] An imaging device may include one or more lenses. A lens may be included in a device as a single lens or an array or plurality of lenses (e.g., compound lens; a lens array may include about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more lenses). A lens can be constructed from any suitable material for transmitting light, and sometimes may be constructed from glass and/or a polymer, for example. A lens may be of a suitable geometry for transmitting light, and nonlimiting examples of lenses include biconvex (double convex, convex), equiconvex, biconcave (concave), plano-convex, plano-concave, convex-concave (meniscus). A lens in a device sometimes focuses light. A lens sometimes focuses light on an image plane of a light filter , and sometimes a lens focuses light on an image plane of a photodetector. A lens sometimes magnifies an image, such as an image transmitted from an illumination region. A lens in some embodiments demagnifies an image, such as image transmitted from a light filter to a photodetector. Magnification or demagnification can be at any suitable level, and sometimes may be about 2* to about 1,000* (e.g., about 10*, 50*, 100*, 200*, 300*, 400*, 500*, 600*, 700*, 800*, 900* magnification or demagnification). A lens sometimes receives light from a channel and focuses that light on an image plane of a light filter . A lens sometimes receives light from a light filter and focuses the light on an image plane of a sensor surface.

Confocal Microscopy

[0108] In some aspects disclosed here in, is a confocal microscope that has the imaging system as described herein. Such a confocal microscope would have the ability to resolve multiple different transmitted and scattered signals simultaneously. It may be capable of real time observations, so making it possible to provide a complex determination of size, structure, life cycle, confirmation, identification, or other features of particles.

[0109] According to the present disclosure, the confocal microscope may comprise a light filter that includes a pinhole filter. In the confocal microscope of the invention, light emitted from a source passes through the particle and then through an array of mirrors, lenses, and light filters. Light reflected from a sample position varying per wavelength may be focused onto an array of pinholes through the longitudinal chromatic aberration-producing optical element and objective to form an image thereon. The light may be reciprocally propagated from the pinholes to the sample, and from the sample to the pinholes, and so the position of the image formed on the pinholes may be invariable irrespective of wavelength. The light passing through the pinholes may be guided to an observation side, so that images on the sample position varying in the optical axis direction can simultaneously be observed in separate colors. Since the system is a confocal optical system, a blurred image at each wavelength may be cut off by the pinholes so that the sample can be observed with high resolving power and high contrast and at a great focal depth. In addition, sample information in the optical axis direction can be learned per image color.

Flow Cytometry and Cell Sorting

[0110] Embodiments disclosed herein relate generally to systems, apparatuses, and methods for flow cytometry and fluorescent activated cell sorting and, in some embodiments, to systems, apparatuses, and methods that encompass microfluidics-based flow cytometry and fluorescent activated cell sorting (FACS), optionally in combination with one or more subassemblies disclosed therein.

[OHl] Traditional cell sorters like the FACS Aria (BD) use pressure pumps with complicated fluidic lines not meant to be disposable for every experiment. Users of traditional cell sorters usually perform rigorous washing steps in between experiments to avoid cross contamination. Microfluidic based cell sorters like the Tyto Cell Sorter (Miltenyi Biotec) or the On-chip Sort (On-chip Biotechnologies) use pressure or syringe pumps to have a consistent flow rate for sorting; however, these pumps may be more expensive.

[0112] In some embodiments, use of a peristaltic pump for pumping fluid into disposable microfluidic flow cells and fluidics as disclosed herein can simplify cleaning and reduce the possibility of cross-contamination. Peristaltic pumps may be affordable and can allow for ease of replacement of any fluidic line(s) that interact with the sample fluid. Further, peristaltic pumps can be relatively more compact than existing pressure pumps, making them suitable for relatively inexpensive instruments that may be within the budgets of most labs.

[0113] In some embodiments, the use of peristaltic pumps further permits for determination of particle concentration in the sample fluid and/or the fluid in which the sorted particles may be present via measurement of liquid volume based on the speed and duration of peristaltic pump action. In this manner, volume measurements may be afforded in a system with a disposable cartridge but without the need for in-line flowmeters or other similar devices. In some embodiments, the sample fluid flow rate can be about 1 pl/min, about 5 pl/min, about 10 pl/min, about 50 pl/min, about 100 pl/min, about 200 pl/min, about 500 pl/min, about 900 pl/min, about 990 pl/min, about 1000 pl/min, including all values and sub ranges in between. In some embodiments, the sheath buffer flow rate can be about 1 pl/min, about 5 pl/min, about 10 pl/min, about 50 pl/min, about 100 pl/min, about 200 pl/min, about 500 pl/min, about 900 pl/min, about 990 pl/min, about 1000 pl/min, including all values and sub ranges in between. For example, the flow rate can be about 24 pl/min for the sample fluid and about 160 pl/min for the sheath buffer.

[0114] The sample fluid/particles or cells therein may be analyzed in substantially real-time as they pass through the interrogation area (e.g., where they interact with the laser beam). In some embodiments the particle concentration after sorting (i.e., of the sorted particle) can be calculated based on sample fluid flow rate, the sheath buffer flow rate, the number of sorted particles, and timing of sorting.

[0115] Conventional flow cytometers use a high-speed analog-to-digital converter (ADC) to sample incoming analog detection signals from commercially available photo detectors. The signals from the photo detectors may represent different particle characteristics. In some embodiments, these characteristics can include forward-scatter, side-scatter, back-scatter, fluorescence emission wavelength, and fluorescence intensity at different excitation wavelengths. After data processing, these signals can provide various types of information, such as enumeration of total particles, enumeration of each sub-population of particles, particle velocity, system detection time, and single- or multiple-particle detection, and/or the like. A user can generate a variety of plots based on the above information and create a “gate” to identify a “target particle”. Afterward, the computer processer typically performs a point-by- point comparison of each new particle value with one or more user-defined gates to make a sorting decision. Because the user-defined gate or gates can have a random shape drawn in a software graphical user interface, and there can be multiple gates with or without dependencies, algorithmic processing can place high demands on the computer processor.

[0116] Also provided here is a method for simultaneously collecting flow cytometry data and imaging data. In some cases, after passing through a collection lens, the initial light path or optical signal, ranging from 0° to roughly 10°, may be directed to the first detector, forming the smallest small angle scatter. It is referred to as a small angle scatter image, forward scatter image, forward scatter brightfield image, brightfield image, or light loss image (BD).

[0117] In some case, after passing through a collection lens, the light of the second path or second optical signal from about 10° to about 30° may be allowed to reach a second detector and constitutes the largest small angle scatter. It is commonly known as a darkfield image, small angle scatter darkfield image, or forward scatter darkfield image.

[0118] In some cases, one could segment the scatter angles into multiple angle-ranges, creating a multitude of data and/or images (for example, 0°+/- 5°, 5°-10°, 10°-15°, etc.). This could serve as a form of angular spectroscopy that utilizes scattered light angles. Depending on the collected light angles, the numerical aperture of the collection lens, and the design of the optical path, the resulting images may be of either bright- or dark-field types. Different angles could define various characteristics of the cell, which is vital in generating data to describe and differentiate cells without the need for exogenous labels.

Computer systems

[0119] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smart phone or any other suitable portable or fixed electronic device.

[0120] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible format.

[0121] Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

[0122] The various methods or processes (e.g., of designing and making the coupling structures and diffractive optical elements disclosed above) outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. Additionally, such software may be written using any of a number of suitable programming languages and/or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

[0123] In this respect, various inventive concepts may be embodied as a computer readable storage medium (or multiple computer readable storage media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other non-transitory medium or tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the invention discussed above. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the present invention as discussed above.

[0124] The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of embodiments as discussed above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the present invention need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present invention.

[0125] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0126] Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

[0127] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. EXAMPLES

[0128] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Testing Aperture Sizes for Brightfield Imaging

[0129] A system was configured so that a 488 nm laser was scanned through a HEK cell attached to a 1 pm bead in a flow cytometer. After the laser was passed through the flow cell and the collection objective, the light beam was split and focused into a PMT through a light filter. The light filter was in the configuration of a resizable aperture, which allowed the light in the center of the aperture through to the PMT, while any light striking the light filter outside of the inner circle did not reach the PMT. This was performed in order to optimize the size of the light filter that would preserve transmitted light data for image generation while filtering out the scattered light to decrease noise and interference.

[0130] The diameter of the aperture was tested at 2 mm, 4 mm, 5.5 mm, 6.5 mm, 7.5 mm, and 10 mm. The results of the test are displayed in FIG. 5. In order to better understand the behavior of the aperture, the same test was also performed for the imaging of 10 pm rainbow beads with the 2 mm, 5.5 mm, and 10 mm diameter aperture. The results are shown in FIG. 6. The results showed that for a 488 nm transmission signal cell images appears differently at small aperture (2mm) and large aperture (10 mm). Cell image contrast plateaus at the aperture size of 5.5-6.5 mm and the optimal aperture size was found to be 5.5-6.5 mm for the system used to perform the experiment.

Example 2: Horizontal Bar Light filter

[0131] A system was configured so that a 488 nm laser and a 561 nm laser was scanned through a 10 pm rainbow bead in a flow cytometer. After the lasers were passed through the flow cell and the collection objective, the light beam was split by mirrors in order to separate the light from each of the lasers and focused each beam separately into a two different PMT through two different light filters. The light filter for the 561 nm laser signal was in the configuration of a rectangular bar blocking the center of the objective, such as exemplified by FIG. 7A. The same experiment was repeated using a second light filter in which the horizontal bar was supplemented with filters disposed along the outer edges of the objective such that light was only able to pass through the light filter in two horizontal bars along the top and bottom of the horizontal bar blocker. This light filter is exemplified by FIG. 7B. [0132] The results of the experiment using the light filter of FIG. 7A is shown in FIG. 8 which shows the 488 transmitted light signal and the associated 561 forward scatter signal after passing through the light filter. The results of the experiment using the light filter of FIG. 7B is shown in FIG. 9 which shows the 488 transmitted light signal and the associated 561 forward scatter signal after passing through the light filter.

[0133] The experiment was then repeated again using HEK cells for imaging instead of the rainbow beads. The results of the experiment using the light filter of FIG. 7A is shown in FIG. 10 which shows the 488 transmitted light signal and the associated 561 forward scatter signal after passing through the light filter. The results of the experiment using the light filter of FIG. 7B is shown in FIG. 11 which shows the 488 transmitted light signal and the associated 561 forward scatter signal after passing through the light filter.

Example 3: Annulus Shaped Light Filter

[0134] A new light filter for the 561 forward scatter signal was designed in order to better isolate and define the forward scatter signal. Examples of the new shape, which comprises a circular ring that allows light to pass through the filter, are shown in FIG. 12.

[0135] The tests for the ability to image various targets was repeated was with the prior experiments. The experiment was conducted with 10 pm rainbow beads, HEK239 cells fixed to 1 pm Envy Green beads. The results of the experiments are displayed in FIG. 13 and FIG. 15, respectively. The correlation coefficient of the signals between the 488 nm transmission signal and the 561 forward scatter transmission signal was calculated for both the rainbow beads and the fixed HEK cells in order to assess the quality of the image generated from the annulus shape forward scatter light filter. The results are shown in FIG. 14 and FIG. 16 respectively. The results demonstrated that the annulus shape obscuration disk design returned complete forward scatter images for both beads and cells both rainbow beads and HEK cell images had high correlation between FSC and transmission channel.

Example 4: Annulus Shaped Light filter Further Testing

[0136] Using the configuration of the last experiment, further experiments were performed on different types of samples to assess the performance of the light filter across a range of applications. The samples tested were wild-type live HEK293 cells, live GFP HEK293 cell, VeriCell human leukocytes, GFP algae, GFP algae treated with antibodies, and wild type algae. The results are shown in FIG. 17, FIG. 19, FIG. 21, FIG. 23, FIG. 25, and FIG. 27 respectively. The correlation coefficient of the signals between the 488 nm transmission signal and the 561 forward scatter transmission signal was calculated for all of the experiments as well with the results shown in FIG. 18, FIG. 20, FIG. 22, FIG. 24, FIG. 26, and FIG. 28 respectively.

[0137] The results showed that the 488 transmission signal and 561 forward scatter signal had different appearance on different cell type while the inverted 488 transmission signal and 561 forward scatter had high correlation among all cell types. Strong 561 forward scatter signal was observed on the edges of cell object among all types, which may be caused by leakage of transmitted light that is not fully blocked by the light filter.

Example 5: Annulus Shaped Light filter Design Optimization

[0138] In order to find the optimal size of the donut shaped light filter experiments were performed Use the obscuration disk that can fully block the darkfield signal and gradually reduce the obscuration disk size to investigate the impact on the 561 darkfield signal. 10 pm rainbow beads were imaged using the system of the previous experiments. The size of the circular middle blocking region of the light filter was varied by the sizes displayed in Table 1. Trial number 1 was used as a reference as the light filter was designed to completely block the forward scatter signal.

TABLE 1

[0139] The results of the experiments for trials 1-6 are displayed in FIG. 29-FIG. 34 respectively.

Example 6: Forward Detection Light Blocking Disk Design

[0140] A dark field mask blocks the transmitted laser beam, allowing the detector to solely gather light scattered by the sample, thereby generating a dark field image. Conversely, a bright field mask obstructs any light scattered by the sample, capturing only the transmitted laser beam, thus creating a bright field image. [0141] Considering the transmitted beam exhibits a gaussian profile, characterized by long tails, and the scattered light lacks a definite profile, filling the entire aperture, an overlap region arises. This region contains both types of light and is consequently discarded by both the dark field and bright field masks. Given the dark field signal's dimness compared to the bright field, a stronger rejection of the incident beam is required in the dark field detection.

[0142] At the masks' location, the bright field mask diameter is set at approximately the 1/e² diameter of the beam, where the beam's power drops to roughly 13.5% of its peak, confining about 90% of the beam's energy. On the other hand, the dark field mask diameter is set to be four times larger than the beam's 1/e² diameter. The operation of a dark field mask and a bright field mask is depicted in FIG. 35A, FIG. 35B, and FIG. 36.

[0143] In some embodiments, referring to FIG. 37, a length of a horizontal axis may be 10.0 mm, 10.2 mm, 10.4 mm, 10.6 mm, 10.8 mm, 11.0 mm, 11.2 mm, 11.4 mm, 11.6 mm, 11.8 mm, 12 mm, 12.2 mm, 12.4 mm, 12.6 mm, 12.8 mm, or 13 mm, including all values and sub ranges in between. In some embodiments, a length of a horizontal axis may be at least 10.0 mm, at least

10.2 mm, at least 10.4 mm, at least 10.6 mm, at least 10.8 mm, at least 11.0 mm, at least 11.2 mm, at least 11.4 mm, at least 11.6 mm, at least 11.8 mm, at least 12.0 mm, at least 12.2 mm, at least 12.4 mm, at least 12.6 mm, at least 12.8 mm, or at least 13.0 mm. In some embodiments, a length of a horizontal axis may be at most 10 mm, at most 10.2 mm, at most 10.4 mm, at most 10.6 mm, at most 10.8 mm, at most 11.0 mm, at most 11.2 mm, at most 11.4 mm, at most 11.6 mm, at most 11.8 mm, at most 12.0 mm, at most 12.2 mm, at most 12.4 mm, at most 12.6 mm, at most 12.8 mm, or at most 13.0 mm. In some embodiments, a length of a horizontal axis may be about 10.0 mm, about 10.2 mm, about 10.4 mm, about 10.6 mm, about 10.8 mm, about 11.0 mm, about 11.2 mm, about 11.4 mm, about 11.6 mm, about 11.8 mm, about 12.0 mm, about

12.2 mm, about 12.4 mm, about 12.6 mm, about 12.8 mm, or about 13.0 mm.

[0144] In some embodiments, referring to FIG. 37, a length of a vertical axis may be 10.0 mm,

10.2 mm, 10.4 mm, 10.6 mm, 10.8 mm, 11.0 mm, 11.2 mm, 11.4 mm, 11.6 mm, 11.8 mm, 12 mm, 12.2 mm, 12.4 mm, 12.6 mm, 12.8 mm, or 13 mm, including all values and sub ranges in between. In some embodiments, a length of a vertical axis may be at least 10.0 mm, at least 10.2 mm, at least 10.4 mm, at least 10.6 mm, at least 10.8 mm, at least 11.0 mm, at least 11.2 mm, at least 11.4 mm, at least 11.6 mm, at least 11.8 mm, at least 12.0 mm, at least 12.2 mm, at least 12.4 mm, at least 12.6 mm, at least 12.8 mm, or at least 13.0 mm. In some embodiments, a length of a vertical axis may be at most 10 mm, at most 10.2 mm, at most 10.4 mm, at most 10.6 mm, at most 10.8 mm, at most 11.0 mm, at most 11.2 mm, at most 11.4 mm, at most 11.6 mm, at most 11.8 mm, at most 12.0 mm, at most 12.2 mm, at most 12.4 mm, at most 12.6 mm, at most 12.8 mm, or at most 13.0 mm. In some embodiments, a length of a vertical axis may be about 10.0 mm, about 10.2 mm, about 10.4 mm, about 10.6 mm, about 10.8 mm, about 11.0 mm, about 11.2 mm, about 11.4 mm, about 11.6 mm, about 11.8 mm, about 12.0 mm, about

12.2 mm, about 12.4 mm, about 12.6 mm, about 12.8 mm, or about 13.0 mm.

[0145] In some embodiments, referring to FIG. 37, an aperture diameter may be 23.4 mm. In some embodiments, an aperture diameter may be 22.0 mm, 22.2 mm, 22.4 mm, 22.6 mm, 22.8 mm, 23.0 mm, 23.2 mm, 23.4 mm, 23.6 mm, 23.8 mm, 24.0 mm, 24.2 mm, 24.4 mm, 24.6 mm,

24.8 mm, or 25.0 mm, including all values and sub ranges in between. In some embodiments, an aperture diameter may be at least 22.0 mm, at least 22.2 mm, at least 22.4 mm, at least 22.6 mm, at least 22.8 mm, at least 23.0 mm, at least 23.2 mm, at least 23.4 mm, at least 23.6 mm, at least

23.8 mm, at least 24.0 mm, at least 24.2 mm, at least 24.4 mm, at least 24.6 mm, at least 24.8 mm, or at least 25.0 mm. In some embodiments, an aperture diameter may be at most 22.0 mm, at most 22.2 mm, at most 22.4 mm, at most 22.6 mm, at most 22.8 mm, at most 23.0 mm, at most 23.2 mm, at most 23.4 mm, at most 23.6 mm, at most 23.8 mm, at most 24.0 mm, at most

24.2 mm, at most 24.4 mm, at most 24.6 mm, at most 24.8 mm, or at most 25.0 mm. In some embodiments, an aperture diameter may be about 22.0 mm, about 22.2 mm, about 22.4 mm, about 22.6 mm, about 22.8 mm, about 23.0 mm, about 23.2 mm, about 23.4 mm, about 23.6 mm, about 23.8 mm, about 24.0 mm, about 24.2 mm, about 24.4 mm, about 24.6 mm, about

24.8 mm, or about 25.0 mm.

Example 7: Backward Detection Annulus Mirror Design

[0146] FIG. 38A illustrates a diagram of the system with an annulus mirror. An annulus mirror is an optical element containing a small ovular (appears circular when viewed at 45 degrees) mirror (surface A in FIG. 38B) centered on a large piece of clear glass (surfaces B & C in FIG. 38B) The glass is anti -reflection coated to maximize transmission. It functions as the backward scattering spatial filter to block the back reflection light from the lasers. It reflects excitation lasers toward the flow cell, while transmitting fluorescence emission and backscatter from the sample to the detector bank. It also blocks any back reflections of the excitation lasers from the surfaces of the flow cell from entering the detector bank. In some embodiments, a wavelength of the excitation lasers comprises 488 nm ± 5 nm, 561 nm ± 5 nm, or 685 nm ± 5 nm. In some embodiments, a wavelength of the excitation lasers is 488 nm ± 5 nm. In some embodiments, a wavelength of the excitation lasers is 561 nm ± 5 nm. In some embodiments, a wavelength of the excitation lasers is 685 nm ± 5 nm. [0147] In some embodiments, the glass comprises fused silica, a highly transparent and highly pure synthetic silica glass, or synthetic quartz glass substrate (FIG. 39A and 39B).

[0148] In some embodiments, the small mirror (surface A) centered on the substrate may be coated with dichroic coating. In some embodiments, the mirror may reflect more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including all values and sub ranges in between, of the excitation lasers. In some embodiments, the mirror may reflect at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the excitation lasers. In some embodiments, the mirror may reflect at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99% or at most 100% of the excitation lasers. In some embodiments, the mirror may reflect more than about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the excitation lasers. In some embodiments, the mirror may reflect more than 97% of the excitation lasers. [0149] In some embodiments, the small mirror centered on the substrate may transmit more than 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or 98%, including all values and sub ranges in between, of all beams with wavelength in a range of 400-850 nm other than the excitation lasers. In some embodiments, the small mirror centered on the substrate may transmit at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of all beams with wavelength in a range of 400-850 nm other than the excitation lasers. In some embodiments, the small mirror centered on the substrate may transmit at most 80%, at most 82%, at most 84%, at most 86%, at most 88%, at most 90%, at most 92%, at most 94%, at most 96%, at most 98%, or at most 100% of all beams with wavelength in a range of 400-850 nm other than the excitation lasers. In some embodiments, the small mirror centered on the substrate may transmit about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, or about 98% of all beams with wavelength in a range of 400-850 nm other than the excitation lasers. In some embodiments, the small mirror centered on the substrate may transmit more than 90% of all beams with wavelength in a range of 400-850 nm other than the excitation lasers.

[0150] The term “an accuracy of mirror" may refer to the precision of a mirror's surface, particularly in its ability to reflect light without distorting the wavefront. In some embodiments, the small mirror centered on the substrate may have accuracy of lambda (X, wavelength)/20. This means that the deviation of the mirror's surface from the ideal shape is less than or equal to one-twentieth of the wavelength of the light it's designed to reflect. In some embodiments, the small mirror centered on the substrate may have an accuracy of X/10, X/12, X/14, X/16, X/18, X/20, X/22, or X/24, including all values and sub ranges in between. In some embodiments, the small mirror centered on the substrate may have an accuracy of at least X/10, at least X/12, at least X/14, at least X/16, at least X/18, at least X/20, at least X/22, or at least X/24. In some embodiments, the small mirror centered on the substrate may have an accuracy of at most X/10, at most X/12, at most X/14, at most X/16, at most X/18, at most X/20, at most X/22, or at most X/24. In some embodiments, the small mirror centered on the substrate may have an accuracy of about X/10, about X/12, about X/14, about X/16, about X/18, about X/20, about X/22, or about X/24. In some embodiments, the small mirror centered on the substrate may have an accuracy of X/20. [0151] In some embodiments, the mirror centered on the substrate may have surface quality of 10-5 scratch-dig specifications. A scratch-dig specification describes the allowable size of scratches (the first number) and digs (the second number) on the mirror surface. For the scratch number, the value correlates to the width of the scratch, while the dig number corresponds to the diameter of a dig or pit on the surface. So, a 10-5 scratch-dig specification means the surface of the mirror can have no scratches wider than 10 micrometers and no digs larger than 0.5 millimeters in diameter.

[0152] In some embodiments, a width of the mirror centered on the substrate may be 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3.0 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4.0 mm, 4.2 mm,

4.4 mm, 4.6 mm, 4.8 mm, 5.0 mm, including all values and sub ranges in between. In some embodiments, a width of the mirror centered on the substrate may be at least 2 mm, at least 2.2 mm, at least 2.4 mm, at least 2.6 mm, at least 2.8 mm, at least 3.0 mm, at least 3.2 mm, at least

3.4 mm, at least 3.6 mm, at least 3.8 mm, at least 4.0 mm, at least 4.2 mm, at least 4.4 mm, at least 4.6 mm, at least 4.8 mm, or at least 5.0 mm. In some embodiments, a width of the mirror centered on the substrate may be at most 2 mm, at most 2.2 mm, at most 2.4 mm, at most 2.6 mm, at most 2.8 mm, at most 3.0 mm, at most 3.2 mm, at most 3.4 mm, at most 3.6 mm, at most 3.8 mm, at most 4.0 mm, at most 4.2 mm, at most 4.4 mm, at most 4.6 mm, at most 4.8 mm, or at most 5.0 mm. In some embodiments, a width of the mirror centered on the substrate may be about 2 mm, about 2.2 mm, about 2.4 mm, about 2.6 mm, about 2.8 mm, about 3.0 mm, about 3.2 mm, about 3.4 mm, about 3.6 mm, about 3.8 mm, about 4.0 mm, about 4.2 mm, about 4.4 mm, about 4.6 mm, about 4.8 mm, or about 5.0 mm. In some embodiments, a width of the mirror centered on the substrate may be between 2 mm and 5.0 mm, between 2.2 mm and 4.8 mm, between 2.4 mm and 4.6 mm, between 2.6 mm and 4.4 mm, between 2.8 mm and 4.2 mm, between 3.0 mm and 4.0 mm, between 3.2 mm and 4.0 mm, or between 3.7 mm and 3.9 mm. In some embodiments, a width of the mirror centered on the substrate may be 3.89 nm. [0153] In some embodiments, a height of the mirror centered on the substrate may be 1.4 mm,

1.6 mm, 1.8 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3.0 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4.0 mm, including all values and sub ranges in between. In some embodiments, a height of the mirror centered on the substrate may be at least 1.4 mm, at least 1.6 mm, at least 1.8 mm, at least 2.0 mm, at least 2.2 mm, at least 2.4 mm, at least 2.6 mm, at least 2.8 mm, at least 3.0 mm, at least 3.2 mm, at least 3.4 mm, at least 3.6 mm, at least 3.8 mm, or at least 4.0 mm. In some embodiments, a height of the mirror centered on the substrate may be at most 1.4 mm, at most 1.6 mm, at most 1.8 mm, at most 2.0 mm, at most 2.2 mm, at most 2.4 mm, at most

2.6 mm, at most 2.8 mm, at most 3.0 mm, at most 3.2 mm, at most 3.4 mm, at most 3.6 mm, at most 3.8 mm, or at most 4.0 mm. In some embodiments, a height of the mirror centered on the substrate may be about 1.4 mm, about 1.6 mm, about 1.8 mm, about 2.0 mm, about 2.2 mm, about 2.4 mm, about 2.6 mm, about 2.8 mm, about 3.0 mm, about 3.2 mm, about 3.4 mm, about

3.6 mm, about 3.8 mm, or about 4.0 mm. In some embodiments, a height of the mirror centered on the substrate may be between 2 mm and 4.0 mm, between 2.2 mm and 3.8 mm, between 2.4 mm and 3.6 mm, between 2.5 mm and 3.4 mm, between 2.6 mm and 3.2 mm, between 3.4 mm and 3.0 mm, between 2.2 mm and 3.0 mm, or between 2.5 mm and 2.9 mm. In some embodiments, a height of the mirror centered on the substrate may be 2.75 nm. The terms "width" of the mirror and "height" of the mirror are used interchangeably herein to refer to the length of one of the sides of the mirror. In some embodiments, a length of one side of the mirror may be 3.89 mm. In some embodiments, a length of one side of the mirror may be 2.75 mm. In some embodiments, a length of one side of the mirror may be 3.89 mm and a length of the other side of the substrate may be 2.75 nm.

[0154] The annulus mirror may be placed at 45° respect to the incident laser light. In some instances, the angle between the annulus mirror and the incident laser light is 30°, 35°, 40°, 45°, 50°, 55°, or 60°, including all values and sub ranges in between. In some instances, the angle between the annulus mirror and the incident laser light may be about 30°, about 35°, about 40°, about 45°, about 50°, about 55°, or about 60°. In some instances, the angle between the annulus mirror and the incident laser light may be at least 30°, at least 35°, at least 40°, at least 45°, at least 50°, at least 55°, or at least 60°. In some instances, the angle between the annulus mirror and the incident laser light may be at most 30°, at most 35°, at most 40°, at most 45°, at most 50°, at most 55°, or at most 60°. In some instances, the angle between the annulus mirror and the incident laser light may be between 30° and 60°, between 35° and 55°, between 40° and 50°, or between 35° and 45°. In some instances, the angle between the annulus mirror and the incident laser light may be 30°, 35°, 40°, 45°, 50°, 55°, or 60°. In some instances, the angle between the annulus mirror and the incident laser light is 45°.

[0155] In some embodiments, the non- mirror surfaces of the substrate (surfaces B and C in FIG. x) may be coated with an anti -reflective coating.

[0156] In some embodiments, the non- mirror surfaces of the substrate may transmit more than 90%, 92%, 94%, 96%, 98%, or 99%, including all values and sub ranges in between, of the light of wavelength of 400-850 nm. In some embodiments, the non- mirror surfaces of the substrate may transmit at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the light of wavelength of 400-850 nm. In some embodiments, the non- mirror surfaces of the substrate may transmit at most 90%, at most 92%, at most 94%, at most 96%, at most 98%, or at most 99% of the light of wavelength of 400-850 nm. In some embodiments, the non- mirror surfaces of the substrate may transmit about 90%, about 92%, about 94%, about 96%, about 98%, or about 99% of the light of wavelength of 400-850 nm. In some embodiments, the nonmirror surfaces of the substrate may transmit more than 99% of the light of wavelength of 400- 850 nm.

[0157] In some embodiments, the non- mirror surfaces of the substrate may have an accuracy of X/2, X/4, X/6, X/8, or X/10, including all values and sub ranges in between. In some embodiments, the non- mirror surfaces of the substrate may have an accuracy of at least X/2, at least X/4, at least X/6, at least X/8, or at least X/10. In some embodiments, the non- mirror surfaces of the substrate may have an accuracy of at most X/2, at most X/4, at most X/6, at most X/8, or at most X/10. In some embodiments, the non- mirror surfaces of the substrate may have an accuracy of about X/2, about X/4, about X/6, about X/8, or about X/10. In some embodiments, the non- mirror surfaces of the substrate may have an accuracy of X/4.

[0158] In some embodiments, the non- mirror surfaces of the substrate may have surface quality of 40-20 scratch-dig specifications. So, the non- mirror surfaces of the substrate can have no scratches wider than 40 micrometers and no digs larger than 0.2 millimeters in diameter.

[0159] In some embodiments, a length of one side of the substrate may be 16 mm, 18 mm, 20 mm, 22 mm, 24 mm, 25 mm, 26 mm, 28 mm, 30 mm, 32 mm, 34 mm, 35 mm, 36 mm, 38 mm, 40 mm, 42 mm, 44 mm, or 45 mm, including all values and sub ranges in between. In some embodiments, a length of one side of the substrate may be at least 16 mm, at least 18 mm, at least 20 mm, at least 22 mm, at least 24 mm, at least 25 mm, at least 26 mm, at least 28 mm, at least 30 mm, at least 32 mm, at least 34 mm, at least 35 mm, at least 36 mm, at least 38 mm, at least 40 mm, at least 42 mm, at least 44 mm, or at least 45 mm. In some embodiments, a length of one side of the substrate may be at most 16 mm, at most 18 mm, at most 20 mm, at most 22 mm, at most 24 mm, at most 25 mm, at most 26 mm, at most 28 mm, at most 30 mm, at most 32 mm, at most 34 mm, at most 35 mm, at most 36 mm, at most 38 mm, at most 40 mm, at most 42 mm, at most 44 mm, or at most 45 mm. In some embodiments, a length of one side of the substrate may be about 16 mm, about 18 mm, about 20 mm, about 22 mm, about 24 mm, about 25 mm, about 26 mm, about 28 mm, about 30 mm, about 32 mm, about 34 mm, about 35 mm, about 36 mm, about 38 mm, about 40 mm, about 42 mm, about 44 mm, or about 45 mm. In some embodiments, a length of one side of the substrate may be 35 mm. In some embodiments, a length of one side of the substrate may be 25 mm. In some embodiments, a length of one side of the substrate may be 35 mm and a length of the other side of the substrate may be 25 nm. [0160] In some embodiments, the thickness of the substrate may be 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm, including all values and sub ranges in between. In some embodiments, the thickness of the substrate may be at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, or at least 10 mm. In some embodiments, the thickness of the substrate may be at most 2 mm, at most 3 mm, at most 4 mm, at most 5 mm, at most 6 mm, at most 7 mm, at most 8 mm, at most 9 mm, or at most 10 mm. In some embodiments, the thickness of the substrate may be about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm. In some embodiments, the thickness of the substrate may be between 2 mm and 10 mm, 3 mm and 9 mm, 4 mm and 8 mm, 4 mm and 7 mm, or 3 mm and 6 mm. In some embodiments, the thickness of the substrate may be 5 mm.

[0161] Any stains on the anti -reflection coating can be gently cleaned with acetone.

Claims

CLAIMS What is claimed is:

1. A method for imaging a particle, comprising:

(a) scanning a particle by directing an illuminating light at a region comprising the particle, thereby generating a signal light;

(b) passing the signal light through an optical element;

(c) splitting the signal light into a first optical signal and a second optical signal;

(d) passing the first optical signal through a first light filter, wherein the first light filter selectively allows a portion of the first optical signal through;

(e) passing the second optical signal through a second light filter, wherein the second light filter selectively allows a portion of the second optical signal through; and

(f) separately detecting the first optical signal and the second optical signal.

2. The method of claim 1, wherein the first light filter and the second light filter comprise a blocking element.

3. The method of claim 1 or 2, further comprising creating an image from a composite of the first optical signal and the second optical signal.

4. The method of claim 2, wherein the blocking element is an adjustable aperture.

5. The method of claim 2, wherein the blocking element is a pinhole aperture.

6. The method of claim 2, wherein the blocking element is in a shape chosen from among a circle, a square, a rectangle, an annulus, a triangle, an oval, or a polygon.

7. The method of any one of claims 1 to 6, wherein the blocking element comprises an inner portion and an outer portion.

8. The method of any one of claims 1 to 7, wherein the inner portion comprises a circular shape and the outer portion comprises a circular shape defining a torus region which the first optical signal is able to pass through.

9. The method of any one of claims 1 to 8, wherein step (c) is performed using a partial metal mirror.

10. The method of any one of claims 1 to 9, wherein the illuminating light comprises a lightemitting diode (LED).

11. The method of any one of claims 1 to 9, wherein the illuminating light comprises a laser.

12. The method of any one of claims 1 to 9, wherein the illuminating light comprises at least two lasers.

13. The method of claim 12, wherein a wavelength of the at least two laser comprises 561 nm or 488 nm.

14. The method of any one of claims 1 to 13, wherein the first optical signal comprises a signal light generated by the laser of 561 nm and the second optical signal comprises a signal light generated by the laser of 488 nm.

15. The method of any one of claims 1 to 14, wherein the lens has a numerical aperture (NA) of at least 0.28.

16. The method of any one of claims 1 to 15, wherein a wavelength of the first optical signal and a wavelength of the second optical signal are different.

17. The method of claim 4, wherein a size of the adjustable aperture ranges from 1 mm to 11 mm.

18. The method of any one of claims 1 to 17, wherein the first optical signal comprises a bright field signal.

19. The method of any one of claims 1 to 18, wherein the first optical signal comprises a darkfield signal.

20. The method of any one of claims 1 to 19, wherein the first optical signal comprises a forward scattering signal.

21. The method of claim 20, wherein the first optical signal comprises a 0°+/- 30° forward scattering signal.

22. The method of any one of claims 1 to 19, wherein the first optical signal comprises a back scattering signal.

23. The method of claim 22, wherein the first optical signal comprises a 180°+/- 30° back scattering signal.

24. The method of any one of claims 1 to 23, wherein the second optical signal comprises a brightfield signal.

25. The method of any one of claims 1 to 23, wherein the second optical signal comprises a transmission signal.

26. The method of claim 2, wherein the first light filter, the second light filter, or both are attached to a filter wheel, wherein the filter wheel may be rotated such that the blocking element may be selected from among multiple blocking elements.

27. The method of claim 1, further comprising: splitting the signal light into a nth optical signal; passing the nth optical signal through a nth light filter, wherein the nth light filter selectively allows a portion of the nth optical signal through; detecting the nth optical signal; and creating an image from the composite of the first optical signal, the second optical signal, and the nth optical signal, wherein the at least one of the nth optical signal are chosen from among a 0°+/- 30° forward scattering signal and 180°+/- 30° back scattering signal.

28. The method of any of the previous claims, wherein the particle lacks a fluorescent label.

29. A system for imaging of moving particles, comprising: a particle motion device comprising a substrate configured to allow particles to move along a travel path in a first direction; an optical illumination system configured to scan with a light in a region of the travel path of a particle; an optical detection system optically interfaced with the particle motion device and operable to obtain optical signal data associated with the particle, wherein the optical detection system comprises one or more beam splitters, one or more photodetectors, one or more adjustable apertures, one or more lenses, and one or more light filters positioned between the particle motion device and the one or more photodetectors, wherein the one or more light filters are configured to selectively allow a portion of the optical signal to pass through the light filters and be detected by the one or more photodetectors; and a data processing unit in communication with the optical detection system, wherein the data processing unit comprising a processor is configured to process the optical signal data obtained by the optical detection system and produce label-free, bright field, transmission, scatter, darkfield and fluorescent image data comprising information indicative of the features of the particle, wherein the one or more light filters selectively allow scattered optical signals through.

30. The system of claim 29, wherein the optical illumination system further comprises one or more LEDs.

31. The system of claim 29, wherein the optical illumination system further comprises a laser.

32. The system of claim 29, wherein the optical illumination system further comprises two or more lasers.

33. The system of claim 29 or 32, wherein the optical illumination system is configured to scan the region of the travel path of the particle with the two lasers simultaneously.

34. The system of any one of claims 29 to 33, wherein the adjustable aperture is configured to remove the scatter optical signal.

35. The system of any one of claims 29 to 34, wherein the one or more light filters comprise a blocking element in a shape chosen from among a circle, a square, a rectangle, a triangle, an oval, or a polygon.

36. The system of any one of claims 29 to 35, wherein the blocking element comprises an inner portion and an outer portion.

37. The system of any one of claims 29 to 36, wherein the inner portion comprises a circular shape and the outer portion comprises a circular shape defining an annulus region which the optical signal is able to pass through.

38. The system of any one of claims 29 to 37, wherein at least one of the one or more light filters selectively allows a 0°+/- 30° forward scatter portion of the optical signal through.

39. The system of any one of claims 29 to 37, wherein at least one of the one or more light filters selectively allows a 180°+/- 30° back scatter portion of the optical signal through.

40. The system of any one of claims 29 to 37, wherein the particle lacks a fluorescent label.

41. An image-based particle sorting system, comprising: a particle flow device structured to comprise a substrate, a channel formed on the substrate operable to flow particles along a flow direction to a first region of the channel, and one or more output paths branching from the channel at a second region proximate to the first region in the channel; an imaging system interfaced with the particle flow device and operable to obtain image data associated with a particle when the particle is in the first region during flow through the channel wherein the imaging system comprises one or more light filters that are configured to selectively filter the image data and simultaneously generate a transmission image data and one or more scattered image data; a data processing and control unit in communication with the imaging system, wherein the data processing and control unit comprising a processor is configured to process the transmission image data and the one or more scatter image data obtained by the imaging system to determine one or more properties associated with the particle from the processed image data and to produce a control command based on a comparison of the determined one or more properties with a sorting criteria, wherein the control command is produced during the particle flowing in the channel and is indicative of a sorting decision determined based on one or more particular attributes ascertained from the image signal data that corresponds to the particle; and an actuator operatively coupled to the particle flow device and in communication with the control unit, wherein the actuator is operable to direct the particle into an output path of the one or more output paths based on to the control command,

42. The image-based cell sorting system of claim 41, wherein the particle lacks a fluorescent label.