Compact wavelength detection module

Abstract

Compact wavelength detection module. The compact light detection module includes an image array having a transparent block, a plurality of micromirrors in a row coupled to a first side of the transparent block, and a plurality of filters in a row coupled to a second side of the transparent block opposite the first side. Each filter of the plurality of filters reflects light to one micromirror of the plurality of micromirrors and transmits light of a different wavelength range, and each micromirror of the plurality of micromirrors reflects light to one filter of the plurality of filters, such that incident light incident to the image array zigzags back and forth between successive filters of the plurality of filters and successive micromirrors of the plurality of micromirrors. A radius of curvature of each micromirror of the plurality of micromirrors images a fiber aperture onto an odd filter and collimates a beam on an even filter.

Description

[0001] This application is a divisional application of the parent application for Invention Patent Application No. 201780058620.2 (International Application No. PCT / US2017 / 043815, filed on July 25, 2017, entitled FLOW CYTOMETER) for the inventors Ming Yan et al.

[0002] Cross Reference to Related Applications

[0003] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 366,580, entitled COMPACT DETECTION MODULE FOR FLOW CYTOMETERS, filed on July 25, 2016, for the inventors Ming Yan et al., which is incorporated by reference herein for all purposes and intents.

[0004] This patent application is also related to Application No. 15 / 498,397, entitled COMPACT MULTI-COLOR FLOW CYTOMETER, filed on April 26, 2017, for David Vrane et al., which describes a flow cytometer that can use a compact detection module, and is incorporated by reference herein for all purposes and intents. TECHNICAL FIELD

[0005] Embodiments of the present invention relate generally to detection modules for flow cytometers. BACKGROUND

[0006] Flow cytometers typically have a viewing aperture that is illuminated by one or more lasers. Laser light from the one or more lasers hits various fluorescently labeled microparticles that pass through the aperture. The fluorescently labeled microparticles are typically various biological cells in a sample that are labeled with different flourochromes (fluorescent dyes), and the cells can be analyzed to obtain information about the sample that can be extrapolated to the whole. One or more optical detectors in the flow cytometer are used to sense the fluorescence (fluorescent light) emitted from the fluorescently labeled microparticles that pass through the aperture that are hit by the laser light from the one or more lasers.

[0007] One or more different optical filters can be placed before the fluorescent light emitted from the fluorescently labeled microparticles reaches each detector. The optical filters are placed in the emitted fluorescent light path so that each detector sees only a particular bandwidth of light associated with the expected fluorescence of a fluorochrome. That is, the bandwidth of any given optical filter utilizes a peak in the emission spectrum of a particular fluorescent dye. In this way, the collective signal from the detectors for any given microparticle indicates the type of fluorochrome or fluorochromes attached to the microparticle. The signal detected by the detectors from the emitted fluorescent light allows for rapid and comprehensive cell classification of the various microparticles in the sample.

[0008] However, the emission spectra can overlap between dyes. This limits the number of different fluorochromes that can be simultaneously detected on a given microparticle by a single laser and detector. Because the emission bandwidth is typically in a wavelength range of 30 nanometers (nm) to 60 nm, conventional flow cytometers can typically detect no more than four or five fluorochromes per laser line. Increasing the number of lasers provides a beneficial, but expensive, method to increase the number of fluorochromes that can be simultaneously detected.

[0009] Further complicating the detection of emitted fluorescent light is the fact that many dyes used to color microparticles are excited in laser wavelength ranges that are different from and larger than the typical 30 nm to 60 nm bandwidth range. This can result in signal cross-talk between detectors for different lasers.

[0010] Existing flow cytometry fluorescence detection systems limit divergence by increasing the focal length of the collimating lens. However, depending on the size of the final image required, this results in a larger diameter beam, which limits the number of detectors, such as six detectors. In these existing flow cytometry systems, the final image size is constrained by optical aberrations when imaging large size optics (such as 800 micrometers (micro-meter or micron) (pm)) and wideband light (e.g., wavelengths of 400 nm - 800 nm) are collimated to a set of detectors with a diameter less than 3 millimeters (mm).

[0011] In another flow cytometry system, a spherical micromirror is utilized to reimage the incoming light for each detector in a chain of detectors that are in a row of detectors. The reimaging avoids the divergence collimated light problem of the flow cytometry system described above. However, the number of detectors is limited by the aberrations introduced by the reflections from the spherical micromirror. As the image size increases along the chain of detectors, large area detectors are required to increase the number of detector channels along the row in the chain of detectors, resulting in a large and expensive flow cytometer.

[0012] The resolution of the emission spectrum is also important for increasing the number of detectable fluorochromes. A detector array can be used to identify fluorochromes based on the aggregate emission signature across multiple wavelengths, thereby increasing the number of detectable fluorochromes. In essence, the entire fluorescent signal is dispersed into a detector array by a diffraction grating or prism. In this way, the entire emission spectrum is discretized across multiple detectors. Spectral unmixing can be used to calculate the contribution of known individual fluorochrome spectra to the total aggregate signal. However, this method for increasing the number of detectable fluorochromes has two major limitations.

[0013] The continuous linear nature of the dispersive component / detector array does not allow for the bandwidth to be adjusted to take advantage of the true nature of the fluorochrome spectra. Thus, the identification of a wider bandwidth favors longer wavelength fluorochromes, while neglecting the details of shorter wavelength fluorochromes with compressed spectra. Furthermore, scattered light from other lasers, if present, is inevitably collected by the detector array. This scattered light compromises the fluorescent signal that the detector is meant to detect.

[0014] Thus, there is a need for further improvements in flow cytometry to increase the number of detectable fluorochromes and better analyze fluorochrome-labeled microparticles in a sample. SUMMARY

[0015] A flow cytometer comprising: a compact light detection module comprising a first image array having a transparent block, a plurality of micromirrors in a row coupled to a first side of the transparent block, and a plurality of optical filters in a row coupled to a second side of the transparent block opposite the first side, wherein each of the plurality of optical filters reflects light to one of the plurality of micromirrors and transmits light of a different wavelength range, and each of the plurality of micromirrors reflects light to one of the plurality of optical filters, such that incident light incident to the image array zigzags back and forth between successive ones of the plurality of optical filters and the plurality of micromirrors, wherein a radius of curvature of each of the plurality of micromirrors directs light onto even ones of the plurality of optical filters along the row and reimages light onto odd ones of the plurality of optical filters. BRIEF DESCRIPTION OF DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color ink. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0017] Figure 1 is a block diagram of a detection module of a flow cytometer system.

[0018] Figure 2A This is a schematic diagram of an image array with continuous channel re-imaging.

[0019] Figure 2B This is a schematic diagram of a 1f image array with alternating channel re-imaging.

[0020] Figure 3 This is a schematic diagram of a compact detection module that includes a 16-channel 1f image array for modular flow cytometry systems.

[0021] Figure 4A This is a magnified image of fluorescence departing at different points along the diameter of the optical fiber.

[0022] Figure 4B This is an enlarged view of a detector used to convert optical signals into electrical signals.

[0023] Figure 5 This is a magnified view of a portion of the 1f image array.

[0024] Figure 6 This is a diagram of the detection module in a modular flow cytometer system with a 16-channel 1f image array and 16 detector channels.

[0025] Figures 7A-7C These are different schematic views of the detection module in a modular flow cytometer system, which has a pair of 8-channel 1f image arrays and a pair of 8 detector channels.

[0026] Figure 8 It is a stereoscopic image of a 1f image array with spherical micromirrors.

[0027] Figures 9A-9B yes Figure 8 A cross-sectional view of the 1f image array.

[0028] Figure 10 It is a stereoscopic image of a 1f image array with concave rectangular micromirrors.

[0029] Figure 11 yes Figure 10 A top-side view of a portion of the 1f image array.

[0030] Figure 12 It is a mounting block for adjacent imaging blocks and for... Figure 6 and Figures 7A-7C A three-dimensional view of a low-cost, thin-shape detector module.

[0031] Figure 13 This is a top view of the optical plate assembly in a modular flow cytometry system. Detailed Implementation

[0032] In the following detailed description of implementations, numerous specific details are set forth in order to provide a thorough understanding. However, it will be apparent to one skilled in the art that the present implementations can be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present implementations.

[0033] Embodiments of the present invention include methods, apparatuses, and systems for flow cytometers with compact, highly multiplexed detection modules.

[0034] A flow cytometer with a compact detection module for fluorescence is disclosed, with increased number of detectors compared to existing flow cytometers, with minimal image size. Each detector module is fed by at least one laser. Multiple lasers can be supported by multiple detector arrays in a compact fashion. The number of detectors can be increased by careful control of incremental aberrations in the detector array as light is transmitted through the detection chain. The compact size of the compact detection module is achieved by a reduced distance between the micromirrors and the optical filters and careful downscaling, thereby minimizing image degradation along the rows or chains of micromirrors and optical filters in the imaging array.

[0035] The existing limitations can be overcome by using an optical system with adjustable progression of multiple individual detectors and filter bandwidths. The optical system enables optimal resolution of the spectra of both long and short wavelength dyes used to label microparticles analyzed by the flow cytometer, with a concentration of spectral configuration points. Fluorescence from excited microparticles is imaged through an objective lens with a high numerical aperture (NA) into a multimode optical fiber. The broadband fluorescence exiting the multimode optical fiber is collimated and then coupled into (imaged into) multiple detectors. Collimation of the broadband fluorescence exiting the multimode optical fiber is a challenge.

[0036] In the following, reference is made to Figure 1 , showing a functional block diagram of a detection module 100 of a flow cytometer. An advanced flow cytometer can include multiple detection modules. The detection module 100 is a wavelength de-multiplexing system. The detection module 100 continuously reflects and reimages fluorescence output 101A from an optical fiber 102 in an image array 106. The image array 106 is a mechanical image array, which includes a plurality of mirrors 110A-110E and a corresponding plurality of long-pass dichroic filters 112A-112E suspended in the air by mechanical mounts. Typically, a dichroic filter is an accurate color filter used to selectively pass light in one range of wavelengths of colored light while reflecting other wavelengths of colored light. Alternatively, the plurality of long-pass dichroic filters 112A-112E can be bandpass filters.

[0037] Image array 106 is capable of reflectively re-imaging a fiber spot N times (where N is greater than 2) while maintaining the optical quality of the fiber spot at the end of the image array. Re-imaging is the function of re-creating the original image with some aberration (reimage) at the surface of each of the dichroic filters 112A-112E, for example. Detection module 100 also includes a plurality of detector channels 113A-113E, which each include a plurality of objective lenses 116A-116E and a plurality of detectors 118A-118E, all in optical communication. Optionally, the plurality of detector channels 113A-113E can also each include a plurality of bandpass filters 114A-114E to ensure that the plurality of detectors detect each desired wavelength range.

[0038] In image array 106, light 101A incident on first mirror 110A is reflected by the mirror as reflected light 103A. Reflected light 103A from first mirror 110A passes through air and is incident on first long pass dichroic filter 112A. Light 103A from first mirror 110A is split by long pass dichroic filter 112A into a continuous light portion 101B and a pass or transmitted light portion 105A. Continuous light portion 101B passes through air and is incident on the next mirror in the series, mirror 110B. Transmitted light portion 105A is coupled into first bandpass filter 114A of first detector channel 113A. Transmitted light portion 105A is cleaned up by bandpass filter 114A and then coupled by objective lens 116A into optical detector 118A. The process is repeated for each stage (detector channel) in image array 106.

[0039] Image array 106 includes five stages that re-image five times on each long pass dichroic filter. It is still desirable to include a larger number of detectors. However, after more than 5 re-imaging passes, the beam distortion through the mirrors can accumulate to the point where the image quality at the last dichroic filter becomes highly degraded. To make the zigzag back and forth configuration between the mirrors and dichroic filters in image array 106 work properly with a larger number of detectors, it is desirable to minimize the image degradation along the optical path.

[0040] Minimizing image degradation in a detection module can be achieved with two mechanisms. Image degradation can be reduced if the bending power of each mirror in the image array is reduced (e.g., by a factor of two - half the bending power). Image degradation can be further reduced if the number of times the beam is re-imaged in the image array is reduced (e.g., by a factor of two - half the number of times).

[0041] Reducing the angle of curvature at the mirror reduces all types of aberrations. Since the aberrations increase non-linearly with the angle of curvature, the improvement obtained by switching from a "fast focusing mirror" to a "slow focusing mirror" is significantly better than 2X, allowing the image quality to be maintained through more reflections. By reducing the number of times the beam is re-imaged in the image array, the image quality incident on the last detector in the chain of detectors can be further improved. Instead of being re-imaged on each dichroic filter for each detector channel, the incident light can be re-imaged every other dichroic filter and detector channel (e.g., odd detector channels).

[0042] Reference will now be made to Figures 2A-2B to describe image arrays 106A-106B that provide an improvement in image quality by utilizing a transparent block with micro-mirrors of different radii of curvature. Typically, an image array is composed of a micro-mirror array and an opposing bandpass and / or dichroic filter array for each detection channel. In each case, the thickness L of the transparent block between the serial chain or row of micro-mirrors M(n), M(n)' on one side and the serial chain or row of dichroic filters D(n) on the opposite side is the same. However, the focal length f of the micro-mirrors M(n) and M(n)' in Figure 2A Figure 2B is different in image arrays 106A-106B.

[0043] Figure 2B The focal length f of the micro-mirrors M(n)' in Figure 2A is L, while the focal length f of the micro-mirrors M(n) in is half of L. The larger focal length of the micro-mirrors M(n)' in image array 106B reduces the angle of curvature and aberrations in the imaging along the mirror serial chain. Also, image array 106B is a If image array that has a thickness L of the given transparent block and a focal length of the micro-mirrors M(n)' such that re-imaging occurs on odd dichroic filters (e.g., dichroic filters D(3), D(5), D(7), to D(n)).

[0044] Figure 2A In Figure 2B , the spot sizes (areas) of the light spots A(l) to A(n) are on the dichroic filters D(l) to D(N), respectively. Spot A(0) is the fiber aperture of the multi-mode fiber from which the fluorescence is input into each array 106A-106B. In Figure 2A and Figure 2B , the fiber aperture can be considered to be infinitesimally small to illustrate the image conjugate properties of both designs.

[0045] Figure 2A ​An image array 106A is illustrated having a plurality of micromirrors M(l) through M(N) and a plurality of long pass dichroic filters D(l) through D(N), where N is an integer value greater than one, representing the number of detector channels. Light focused at a spot A(l) at the dichroic filter D(l) is re-imaged by reflection through the micromirror M(l) to focus the light at a spot A(2) at the dichroic filter D(2) by reflection through the micromirror M(l). This process is repeated for each of the micromirrors M(2) through M(N) along a serial chain or row. The image array 106A is a 2f image array.

[0046] Figure 2B A If design is illustrated for an image array 106B having a plurality of micromirrors M(l)' through M(N)' and a plurality of long pass dichroic filters D(l) through D(N). The micromirrors M(l)' through M(N)' have different radii of curvature than the micromirrors M(l) through M(N). The If image array 106B provides an image quality improvement over the 2f image array 106A at the detectors (e.g., detectors 118A-118E in FIG. 1 IB). Figure 1

[0047] The chain of micromirrors M(l)' through M(N)' in the If image array 106B is designed to relay the image along the chain by the properties of the telescope optical system. For example, by the telescope effect of the micromirrors M(l)' and M(2)', light focused at a spot A(l) at the dichroic filter D(l) is imaged to a spot A(3) at the dichroic filter D(3). The even spots (spot A(2)) are intermediate points in the collimated space.

[0048] The plurality of long pass dichroic filters D(l) through D(N) can alternatively be passband or bandpass filters, or include both a dichroic filter and a bandpass filter combined together to ensure that light of a limited wavelength range passes through. The dichroic filters use thin film interference principles, and can also be referred to as interference filters. For each channel, the bandpass or passband filter is tuned to pass light of a different selected wavelength range (passband) to each detector, and reflect the remaining wavelengths of light back to the micromirrors in the micromirror array.

[0049] To provide a compact detector module, the If image array 106B is formed of a solid transparent material, as further described with reference to Figure 8 and 10 The solid transparent material for the transparent imaging block of the image array can be a transparent glass or a transparent plastic, for example, with the mirrors and dichroic filters formed in or on the transparent material.

[0050] ​The If design of image array 106B and the 2f design of image array 106A can be compared under the condition of the same thickness of solid transparent material, the same pitch, and the same angle of incidence. The path distance between adjacent micromirrors in image array 106A and image array 106B is similar. In this case, the path distance is the physical distance in which the refractive index of the material is considered, rather than the conventional path length. However, the focal length of the micromirrors in the 2f image array 106A is half (one-half) that of the micromirrors in image array 106B. In other words, due to the difference in the radius of curvature in the micromirrors, the actual focal length of the micromirrors in the If image array 106B is twice that of the micromirrors in the 2f imaging array 106A. The longer focal length reduces the bending force of each reflection, which minimizes the aberrations introduced in the micromirror reflections. Thus, the aberrations in the If image array 106B are improved compared to the aberrations of the 2f image array 106A.

[0051] In the 2f image array system 106A, the fiber aperture A(0) is imaged to a spot A(l) at the dichroic filter D(l). The image at spot A(2) is a re-imaging of spot A(l) reflected by micromirror M(l). Continuing through the zigzag optical path in array 106A, each spot A(n) at each dichroic filter D(n) is imaged by the next micromirror M(n) to the next spot A(n+1) at the next dichroic filter D(n+1). In this configuration, the path distance between spot A(n) and micromirror M(n) is the thickness L of the transparent block, which is twice (two times) the focal length of micromirror M(n).

[0052] In the If image array system 106B, light from fiber aperture A(0) is imaged through the input channel to a spot A(l) at the dichroic filter D(l). In image array system 106B, the optical path from micromirror M(l)' to micromirror M(2)' can be considered to form an equivalent telescope with a magnification of 1. In this case, through the telescope of micromirrors M(l)' and M(2)', spot A(l) is imaged to spot A(3), with spot A(2) being an intermediate spot in the collimated space. Adjacent micromirrors M(3)' and M(4)' form another telescope to re-image spot A(3) to spot A(5). Continuing the zigzag path in imaging array 106B, odd spots A(l), A(3), A(5),..., A(2n+1) are all conjugate to each other, while even spots A(2), A(4), A(6),..., A(2n) are intermediate spots in the collimated space. In this configuration, the path distance between spot A(n) and micromirror M(n)' is the thickness L of the transparent block, which is one focal length of micromirror M(n)'.

[0053] Each spot on the filter is formed by a bundle of rays. The angular distribution of the bundle is determined by the numerical aperture (NA) of the input multimode fiber 102 and the input channel of the imaging array of the imaging system. The cone angle at spot A(l) is proportional to the numerical aperture of the multimode fiber 102. If the image magnification m from the fiber aperture A(0) to the spot A(l) is a ratio of 1 to m (where m is greater than 1), the cone angle at spot A(l) is m times smaller than the cone angle at the fiber aperture A(0).

[0054] In the 2f image array system 106A, the image magnification from any spot A(n) to the adjacent spot A(n+1) is equal to 1 for any value of n. Without aberration, the cone angle of light at the dichroic filters D(l) to D(N+1) of the image array system 106A is the same for all detector channels. Figure 2A

[0055] Below, reference is made to Figure 2B The distance between the dichroic filter D(l) and the micromirror M(l)' is L. If the total number of channels N is even in the If image array 106B, the spot A(l) is imaged to spot A(3) by the micromirror M(l)' - M(2)' (considering a 1X telescope), and the spot A(2) at the dichroic filter D(2) is in the collimated space. A portion of the collimated light at spot A(2) is reflected by the dichroic filter D(2) toward the micromirror M(2)'.

[0056] Therefore, the image array 106B has odd detector channels at spots A(l), A(3),..., A(2k-l),..., A(N-l) and even detector channels at spots A(2), A(4),..., A(2k),..., A(N), where 1 < k ≤ (N / 2). Since the spot A(l) is at the front focal point of the micromirror M(l)' and the path distance between adjacent micromirrors is twice the focal length, all odd spots A(l), A(3), A(5),..., A(2n+l) are images of the fiber aperture (as shown in Figure 2B the converging rays of light at odd spots), and all even spots A(2), A(4), A(6),..., A(2n) are in the collimated space (as shown in Figure 2B the parallel rays of light at even spots). Therefore, as shown in Figure 5 the cone angle of odd spots of odd detector channels (odd cone angle CAO) is different from the cone angle of even spots of even detector channels (even cone angle CAE).

[0057] ​In the 1f image array 106B, the center wavelength and passband width of each optical dichroic filter D(l) through D(N) are different from one another. The center wavelength and passband width of each optical dichroic filter are designed to optimize the sampling of the dye fluorescence spectrum to better accurately resolve a large number of different dyes. For example, assume a fluorescence spectrum of optical wavelengths from 400 nm to 800 nm and a sixteen (16) channel detection module to analyze a bandwidth of 25 nm of optical wavelengths each. For example, the first detector channel and the first dichroic filter D(l) can passband and analyze optical wavelengths from 400 nm to 425 nm with a center wavelength of 412.5 nm. Essentially, wavelengths outside of 400 nm to 425 nm are filtered out and not transmitted to the first detector in the first detector channel. The second detector channel and the second dichroic filter D(2) can passband and analyze optical wavelengths from 425 nm to 450 nm with a center wavelength of 437.5 nm, and so on for each detector channel. The last or sixteenth detector channel and the sixteenth dichroic filter D(16) can passband and analyze optical wavelengths from 775 nm to 800 nm with a center wavelength of 787.5 nm.

[0058] The characteristics of the 1f image array 106B allow the initial light signal to propagate into a greater number of detectors than the image array 106A. The 1f image array 106B reduces the off-axis aberrations by reducing the bending force in each mirror reflection. However, in the case where the odd cone angle CAO at the dichroic filter in the odd channels is different from the even cone angle CAE at the dichroic filter in the even channels, the optimal magnification m in the input stage from the fiber aperture A(0) to the light spot A(l) at the dichroic filter D(l) needs to be determined. For a given fiber numerical aperture (NA) and aperture diameter, the magnification m from the fiber aperture A(0) to the light spot A(l) at the dichroic filter D(l) is optimized for both the odd detector channels and the even detector channels in the 1f image array 106B.

[0059] From the perspective of spectral resolution, the performance of the dichroic filter deteriorates as the cone angle of the incident light spot increases. In the 1f image array 106B, the light spot cone angle in the odd channels is different from the light spot cone angle in the even channels. Essentially, the cone angle in the odd channels is determined by the numerical aperture (NA) of the multi-mode fiber and the magnification factor m from the fiber aperture A(0) to the light spot A(l) at the dichroic filter D(l). In contrast, the cone angle of the even channels is determined by the light spot diameter of the light spot in the odd channels.

[0060] In the input channel, assume the image magnification is m from the aperture A(0) at the fiber to the spot A(1) at the bichromatic filter D(1). At the even-numbered detector channel (bichromatic filter D(2k)), the cone angle is proportional to the magnification m. However, at the odd-numbered detector channel (bichromatic filter D(2k-1)), the cone angle is inversely proportional to the magnification m. The larger magnification from the fiber aperture A(0) to the spot A(1) results in a smaller cone angle at the odd-numbered detector channel (bichromatic filter D(2k-1)), but a larger cone angle at the even-numbered detector channel (bichromatic filter D(2k)). In an example implementation with multimode fiber, where NA = 0.12, aperture diameter 600 μm, and micromirror (filter) pitch 5.5 mm, the recommended magnification m is modeled as approximately 2. It is evident that other magnifications m can be determined using different inputs, therefore the embodiments disclosed herein are not limited to a 2X magnification. In the example presented for a 1f image array 106B, both the numerical aperture (NA) and the number of re-images are reduced by a factor of 2, thereby allowing at least four times (4X) more detectors along rows of the same length compared to a 2f image array 106A.

[0061] Figure 3 , Figure 4A – Figure 4B and Figure 5 The diagram illustrates a graph of simulation results for a compact detection module with a 1f image array 106B, an optical fiber with NA = 0.12, an aperture diameter of 600 μm, and a micromirror (filter) pitch of 5.5 m; and example input values ​​for an implementation with a recommended magnification of 2X. Figure 3 , Figure 4A – Figure 4B and Figure 5 The different colors of light shown are merely for clarity to illustrate how light passes through the detector module at different locations.

[0062] Figure 3 The diagram shows one end of the optical fiber 102 that emits fluorescence into the detection system. Near the opposite end of the optical fiber 102 (not shown), a collection objective lens with a high aperture can be used to focus the fluorescence from the aperture and couple it into that opposite end of the fiber. The optical fiber 102 then collects the light from the objective lens and guides it to… Figure 3 The end shown. Near the end. Figure 3 As shown at the end, the system may include a fiber optic numerical aperture converter to reduce the numerical aperture to free space to emit fluorescence to a detector array.

[0063] Below, refer to Figure 3The magnification m in the compact detection module 300 is achieved by the input stage 301. The input stage 301 includes a collimating lens 302, a blocking filter 303, and a focusing lens 304. The magnification m is achieved by adjusting the focal length ratio of the collimating lens 302 and the focusing lens 304. For example, to set the magnification equal to two (m = 2), the focal length of the focusing lens 304 is twice the focal length of the collimating lens 302. The input channel 301 can be considered to also include the input portion of the transparent block in the image array 106B (e.g., wedge shape, block thickness, see Figure 8 ) before reaching the first dichroic D(l).

[0064] The collimating lens 302 receives the light emitted from the optical fiber 102 and collimates the light. The collimated light passes through the blocking filter 303 and is input to the focusing lens 304. The blocking filter 303 is used to clean up the laser light scattered into the collection optical system near the opposite end of the optical fiber 102. The light of the fluorescent spectrum associated with the fluorochromes passes through the blocking filter 303 and into the focusing lens 304. The focusing lens 304 focuses the light of the fluorescent spectrum onto the first dichroic D(l) in the image array 106B to form an image at the light spot A(l). The size (e.g., diameter and area) of the image at the light spot A(l) is magnified m times from the size at the aperture A(0) at the fiber. The positions of the lenses 302, 304 between the end of the optical fiber 102 and the image array 106B can be adjusted.

[0065] The compact detection module 300 also includes sixteen (16) channel If image arrays 106B and sixteen (16) detector channels 313A-313P in communication with the image arrays 106B (see, e.g., Figure 6 ). In alternative embodiments, a pair of eight (8) channel If image arrays (see, e.g., Figure 7A – Figure 7C ) can be used in parallel to relax the imaging requirements of each compact image array. In a flow cytometer, more than one (e.g., three) of these compact image arrays can be used to multiply the number of detector channels to be greater than sixteen (e.g., three times sixteen for forty detector channels), as illustrated by the detector module described with reference to Figure 13 .

[0066] The image array 106B is formed from a solid transparent block material. The sixteen (16) channel If image array 106B includes sixteen (16) dichroic filters D(l) through D(16) on one side of the transparent block and fifteen mirrors M(l) through M(15) on the opposite side. After the last detector channel 313P, the image array does not require a mirror. In addition, the last filter D(16) 314 can not be a dichroic filter; instead, a bandpass filter can be used. In the case of a bandpass filter, the incident light does not need to be further reflected to another mirror or filter.

[0067] Each detector channel 313A-313P (collectively, detector channels 313) in the array or detector includes a focusing lens 316 and a detector 318 (one example of which is shown in FIG. 3B). The detector 318 is packaged in a thin outline (TO) can package 320 to which the focusing lens 316 is coupled or integrated. The focusing lens 316 focuses the fluorescence that passes through the filter onto a small area size of the detector 318. Figure 3

[0068] Below, with reference to Figure 4A , the optical fiber 102 used to transmit the fluorescent signal captured from the image chamber to the detector array is a multi-mode optical fiber. Light exits the end face of the multi-mode optical fiber from each (if not all) of the locations (e.g., locations Xl through X5) on the diameter of the optical fiber. Figure 3 The lenses 303, 304 shown in the input channel focus the light within an aperture A(0) onto a light spot A(l) on the first dichroic filter D(l). Because there is a two times (2X) magnification from the light spot A(0) to the light spot A(l), the light spot size at the aperture A(0) is smaller than the light spot size at the light spot A(l). From Figure 4A The different colors of the light rays emanating from the different locations Xl through X5 within the aperture shown in FIG. 3C are only for clarity to show how the light at the different locations passes through the detector module. As shown in Figure 4A The optical axis 402 extends outward from the center of the circle of the end of the optical fiber 102. The light emanates from the end of the optical fiber 102 at an emission cone angle (CA) 404 relative to the optical axis 402.

[0069] Figure 3 The simulation results for the image array 106B are shown and how the light from different locations in the beam is alternately imaged and collimated through multiple reflections of the mirrors and dichroic filters. While these results show all of the light reflections, the dichroic filters D(n) at any particular location are different and allow the transmission of the light signal according to their respective passbands (only in the last detector channel 313P shown in FIG. 3C). Figure 3

[0070] Below, with reference to​​Figure 3 and Figure 4B In each detector channel 313, the light signal of the desired wavelength range that passes through the dichroic filter D(n) can be focused by lens 316 and detected by a small aperture photosensitive detector 318. An additional bandpass filter 314 can be alternatively or further used in each detector channel. Light of other wavelengths, if any, at the dichroic filter D(n) is reflected along the chain or row of micromirrors to the next micromirror M(n). The dichroic filter D(n) of the row or chain resolves the different ranges of wavelengths of light into the chain of detector channels 313A-313P.

[0071] Figure 5 The magnified view of the optical beam shows how the simulation of the alternating imaging and collimation of the optical beam by the micromirrors and the reflecting surfaces of the dichroic filters. On the odd numbered dichroic filters (e.g., dichroic filters D(7), D(9), and D(11) shown in Figure 5 The light spot is the image of the fiber aperture on the odd numbered dichroic filters (e.g., dichroic filters D(7), D(9), and D(11) shown in Figure 5 On the even numbered dichroic filters (e.g., dichroic filters D(8), D(10), and D(12) shown in Figure 5 The light spots A(8), A(10), and A(12) shown are in the collimated space where the light rays emitted from a point at the fiber aperture become collimated beams. The direction of the beams in the collimated space at each even numbered dichroic filter is slightly different for different points from the fiber aperture.

[0072] In a flow cytometer, one or more linear 16-channel compact wavelength detection modules can be used to detect the fluorescence signals of the light associated with the microparticles. Alternatively or in conjunction, one or more dual 8-channel compact wavelength detection modules can be used in a flow cytometer to detect the fluorescence signals of the light associated with the microparticles.

[0073] Figure 6 and Figures 7A-7C Embodiments of compact wavelength detection modules are illustrated that have the functionality of the 1f image array 106B shown in Figure 2B Embodiments of compact wavelength detection modules are illustrated that have the functionality of the 1f image array 106B shown in Figure 6 A linear 16-channel compact wavelength detection module 600 is illustrated. Figures 7A-7C A dual 8-channel compact wavelength detection module 700 is illustrated.

[0074] In the following, reference is made to Figure 6The linear 16-channel compact wavelength detection module 600 includes an input stage (nosepiece) 601 and a detection module 614 mounted to a base 610. Light is coupled into the input stage (nosepiece) 601 through the optical fiber 102. The input stage (nosepiece) 601 includes a collimating lens 602, a long pass filter 603, a clean optical blocker 604, and a focusing lens 605 mounted to an optical bench. The input stage (nosepiece) 601 sets the magnification m of the initial spot size image A(l) on the first dichroic filter.

[0075] From the input stage 601, light is coupled into the detection module 614. One end of the input stage (nosepiece) 601 is coupled to a transparent optical wedge 607 to receive light from the focusing lens 605. The input stage (nosepiece) 601 and the detection module 614 are coupled to the chassis or base 610 of the flow cytometer to maintain their alignment.

[0076] The detection module 614 includes a 1f image array 608 and a detector / lens array 611. The image array 608 is an embodiment of the image array 106B of Figure 2B and Figure 5 The image array 608 includes a transparent block 680 (see, e.g., the block 806, 1006 of Figure 8 and Figure 10 The image array 608 includes a transparent block 680 (see, e.g., the block 806, 1006 of Figure 3 and Figure 3 The image array 608 includes a transparent block 680 (see, e.g., the block 806, 1006 of

[0077] The light coupled into the image array 608 by the input stage 601 is wavelength resolved into the detectors D1-D16 of the detector / lens array 611. The 16-channel detection module analyzes a range of wavelengths (e.g., 400-800 nm wavelengths).

[0078] To provide better fit to different footprints of test platforms and to provide parallel processing, the linear 16-channel compact wavelength detection module 600 can instead be implemented as a dual 8-channel compact wavelength detection module.

[0079] In the following, reference is made to Figure 7AFIG. 7 shows a top view of a dual detection module 700 having a pair of 8 channel compact wavelength detection modules 714, 715. The compact wavelength detection module 700 includes an input stage (nosepiece) 701 in communication with a first 8 channel detection module 714 and a second 8 channel detection module 715, all of which are mounted in alignment with a base 710. The first 8 channel detection module 714 resolves and analyzes in parallel for a first wavelength range (e.g., 650 nm to 800 nm - red light wavelengths). The second 8 channel detection module 715 resolves and analyzes in parallel for a second wavelength range (e.g., 400 nm to 650 nm - blue light wavelengths).

[0080] Light emitted from the optical fiber 102 is coupled into the input stage (nosepiece) 701. Light from the optical fiber 102 passes through a collimating lens 702 into a long pass dichroic filter 703. The long pass dichroic filter 703 reflects light at the laser excitation wavelength (e.g., less than 400 nm) at a 45 degree angle to a scatter detector (not shown). The side scatter (SSC) light can be focused onto a small aperture detector with a ball lens similar to that described for fluorescence. Fluorescence at the fluorescence spectrum (e.g., 400 nm - 800 nm) passes through the long pass filter 703 and into a second clean-up filter 704. The clean-up filter 704 ensures that no excitation laser light reaches the resolving detection modules 714-715.

[0081] After the clean-up filter 704, the fluorescence is separated into a long wavelength band and a short wavelength band by a long pass filter 705. The long wavelength light (e.g., red light - 650 nm to 800 nm) passes through the long pass filter 705 and is focused by a collimating / focusing lens 706 into the first detection module 714. The long wavelength portion of the light that passes through the long pass filter 705 is resolved by the first detection module 714. The short wavelength light band (e.g., blue light - 400 nm to 650 nm) is reflected by the long pass filter 705 at an angle back into a collimating / focusing lens 713. The collimating / focusing lens 713 focuses the light of the short wavelength band into the second detection module 715. The short wavelength portion reflected by the long pass filter 705 is resolved by the second detection module 715. Alternatively, the filter 705 can be a short pass filter and the short wavelength light passes through the filter and is resolved by the first detection module 714, while the long wavelength light is reflected by the filter and resolved by the second detection module 715.

[0082] Referring to the first detection module 714, the light from the focusing lens 706 enters a 12 degree wedge 707 perpendicularly before imaging onto a first dichroic or bandpass filter 709, and passes through a transparent block of an image array 708 (e.g., a CMOS array) before imaging onto a second dichroic or bandpass filter 710. The second detection module 715 is similar to the first detection module 714, but with the roles of the long and short wavelength bands reversed. Figure 8through the bandpass filter 709 and focused onto a first small area detector D1 in the detector / lens array 711. Light rejected by the bandpass filter 709 is reflected onto a first micromirror M(l) 712 in a plurality of micromirrors M(l) to M(7) in the image array. The first micromirror M(l) 712 collimates and reflects the light back to a second detection module D2, and so on, until the end of the serial chain of micromirrors and detection modules of the transparent block of the image array 708. The second detection module 715 functions similarly to the first detection module 714.

[0083] The reflection through the image array 106B is as described above, with the light being alternately focused and collimated by the 708, 708' in each of the first and second detection modules 714, 715, successively shorter bandpass light passing through the dichroic filter into odd and even detectors 118 in odd and even detector channels, respectively. Thus, the different wavelengths are resolved by the plurality of detectors in each of the first and second detection modules 714, 715.

[0084] For a given fluorescence event, the signal from each detector (e.g., Figure 4B The detectors 318 shown in FIG. 3A, Figures 6-7C The signals from the lenses / detectors D1 to D16 in the block 806) are amplified, digitized, and synchronized by the electronic system to provide a spectral representation of the input fluorescence signal. By minimizing the coupling length of the detectors and amplification circuitry, integrating the detection electronics into the optical module assembly allows for a compact design and lower noise. Figure 4B The detectors 318 shown in FIG. 3A convert an optical signal, such as the input fluorescence signal, into an electrical signal.

[0085] Figure 7B and Figure 7C A right side perspective view and a left side perspective view of a dual detection module 700 with a pair of 8-channel compact wavelength detection modules 714, 715 are respectively illustrated. Each detection module 714, 715 includes a mounting base 720 and a cover 722 to enclose the mounting block 1200 (see Figure 12 ) to which the lenses / detectors 711 in the detector array or chain are mounted. The mounting base 720 and the cover 722 align the components of the image array 708, 708' in the transparent block 806, 1006 with the detector array in each detection module 714, 715. The mounting base 720 of each detection module 714, 715 is coupled to the base 710 by a plurality of fasteners.

[0086] The input stage 701 includes an optical bench 751 having a plurality of filter slots for housing filters 703-705, a plurality of lens slots for housing lenses 702, 706, 713, and one or more optical channels along which light reflects and travels through the filters and lenses. The optical bench 751 is coupled to the base 710 of the detection module 700 to maintain alignment with the detection modules 714-715.

[0087] Referring now to Figure 13 , a top view of an optical board assembly 1300 in a modular flow cytometry system is shown. The optical board assembly 1300 includes a laser system 1370 having three semiconductor lasers 1370A, 1370B, 1370C that direct excitation into a flow cytometry assembly 1308 in which a sample fluid flows with sample particles. The laser system 1370 attempts to direct multiple (e.g., three) laser beams in a collinear fashion toward the flow cytometry assembly 1308. However, the multiple laser beams can be slightly offset from one another. The laser system 1370 includes semiconductor lasers 1370A, 1370B, 1370C at wavelengths of approximately 405 nanometers (nm), 488 nm, and 640 nm, respectively. The output power of the 405 nm semiconductor laser is typically greater than 30 milliwatts (mW); the output power of the 488 nm semiconductor laser is typically greater than 20 mW; and the output power of the 640 nm semiconductor laser is typically greater than 20 mW. Controller electronics control the semiconductor lasers to operate at constant temperature and constant output power.

[0088] An optical system spatially manipulates the optical laser beams 1371A, 1371B, 1371C generated by the semiconductor lasers 1370A, 1370B, 1370C, respectively. The optical system includes lenses, prisms, and turning mirrors to focus the optical laser beams onto a fluid stream carrying bio cells. The focused optical laser beam size is typically focused across the flow stream by 50 micrometers (pm) - 80 pm, and typically focused along the flow flowing in the flow cytometry assembly 1308 by 5 pm - 20 pm. In Figure 13In this embodiment, the optical system includes beam shapers 1330A-1330C that receive laser light 1371A, 1371B, 1371C from semiconductor lasers 1370A-1370C, respectively. The laser light output from the beam shapers 1330A-1330C is coupled into mirrors 1332A-1332C, respectively, to direct laser light 1399A, 1399B, 1399C toward and into the flow cytometry assembly 1308 to target particles (e.g., biological cells) that are stained with a fluorochrome dye. The laser light 1399A, 1399B, 1399C are slightly separated from each other, but enter the flow cytometry assembly 1308 directly and approximately in parallel through the mirrors 1332A-1332C.

[0089] The laser beams 1399A, 1399B, 1399C reach biological cells (particles) in the flow stream in the flow cytometry assembly 1308. The laser beams 1399A, 1399B, 1399C are then scattered by the cells in the flow stream, causing the fluorochrome to fluoresce and produce fluorescence. The forward scatter diode 1314 collects on-axis scattered light. The collection lens 1313 collects off-axis scattered light and fluorescence and directs them together to the dichroic mirror 1310. The dichroic mirror 1310 focuses the off-axis scattered light onto the side scatter diode 1315. The dichroic mirror 1310 focuses the fluorescence onto at least one fiber optic tip 1316. The at least one fiber optic assembly 102 routes the fluorescence to the at least one detector module 600, 700.

[0090] To perform more detailed analysis of a biological sample with different fluorescent dyes and laser wavelengths, multiple fiber optic tips 1316, multiple fiber optic assemblies 102, and multiple detector modules 600, 700 can be used. Three fiber optic tips 1316A, 1316B, 1316C can be disposed in parallel to receive fluorescence, and three fiber optic assemblies 102A, 102B, 102C can be used to direct the fluorescence to three detector modules 600A, 600B, 600C or 700A, 700B, 700C.

[0091] The three fiber tips 1316A, 1316B, 1316C (and the three fiber assemblies 102A, 102B, 102C) are enabled because the three laser beams 1399A, 1399B, 1399C are slightly offset (e.g., not precisely collinear). Thus, the three fiber tips 1316A, 1316B, 1316C can collect beam data from the three laser beams 1399A, 1399B, 1399C, respectively, which have three different wavelengths. The three fiber assemblies 102A, 102B, 102C then direct the light to three different detector modules (e.g., three different detector modules 600A, 600B, 600C or 700A, 700B, 700C).

[0092] Alternatively, the modular flow cytometry system can use one detector module 600, 700 to collect beam data. For example, the three fiber assemblies 102A, 102B, 102C can direct light into one detector module 600, 700, rather than three different detector modules. Separation of the beam data is then performed as a data processing operation, rather than with three different detector modules to separate the beam data. From a physical device perspective, utilizing one detector module can be less complex. However, the data processing operation can be more complex because separation of the beam data requires more data manipulation (e.g., identifying different wavelengths and separating the beam data accordingly).

[0093] Cell geometric features can be classified by analysis of forward and side scatter data. Cells in the fluid stream are labeled with dyes in the visible wavelength range of 400 nm to 900 nm. Upon excitation by the laser, the dyes produce fluorescence, which is collected by the fiber assemblies 102 and routed toward the detector modules 600, 700. The modular flow cytometer system maintains a relatively small size for the optical board assembly via compact semiconductor lasers in the detector modules 600, 700, 11.5X power collection lenses 1313, and compact image arrays.

[0094] The collection lens 1313 contributes to the design of the detector modules 600, 700. The collection lens 1313 has a short focal length of 11.5X power. The collection lens 1313 (objective lens) has a high numerical aperture (NA) of about 1.3 facing the fluorescence emission to capture more photons in the fluorescence emission over a wide range of incident angles. The collection lens 1313 has a low NA of about 0.12 facing the collection fiber 102 to get the fluorescence emission into the fiber over a narrow cone angle. Thus, the collection lens 1313 converts the high NA from one side to a low NA on the opposite side to support the magnification m in the input channel of the detector module 600, 700.

[0095] The collection optical fiber 102 has a core diameter between about 400 pm and 800 pm, and for a core diameter of about 600 pm, the optical fiber NA is about 0.12. The optical fiber output end can be tapered to a core diameter between about 100 pm and 300 pm to control the imaging size onto the receiving photodiode.

[0096] The collection optical fiber 102 input end can also include a lensed optical fiber end to increase the collection NA, allowing the use of a fiber core diameter less than about 400 pm. Because the optical fiber 102 has the flexibility to deliver light anywhere in the flow cytometer system, the use of an optical fiber for fluorescence collection enables the optimization of the location of the receiver assembly and electronics, enabling a compact flow cytometer system.

[0097] To make a low-cost flow cytometer, lower cost components can be introduced. The image array 106B in each detection module 614, 714, 715 is formed from a solid transparent material to provide a reliable, low-cost, and compact detection module. Also, the flow cytometer uses low-cost, off-the-shelf, thin outline (TO) can detectors.

[0098] Below, reference is made to Figure 12 , shown near the transparent blocks 806, 1006 (see Figure 8 – Figure 11 ) are shown mounting blocks 1200 that are coupled together to form the 1f image array 708, 708’ of the base 710 of the compact detector module 700 that is to be mounted to the Figures 7A-7C The mounting blocks 1200 include a plurality of angled curved openings 1201 to accommodate a plurality of TO can lenses / detectors 711. The alignment of the mounting blocks 1200 with the transparent blocks 806, 1006 of the imaging array 708 and the angle of the angled curved openings 1201 are such that light reflected from the micro-mirror 712E can be band-pass filtered by the dichroic filter 709E and coupled to the lens / detector 711E.

[0099] Each TO can lens / detector 711 includes a focusing lens 1211 and a low-cost TO can detector 1212 that are coupled together. The TO can detector 1212 includes a window top and a semiconductor photodetector 1213 inside the TO can package. The semiconductor photodetector 1213 is electrically coupled to a plurality of electrical pins 1214 that extend outside the TO can package to which other electronics of the flow cytometer are electrically coupled. Like the detector 318 shown, Figure 4B Like the detector 318 shown, the semiconductor photodetector 1213 converts an optical signal, such as an incoming fluorescence signal, to an electrical signal on at least one of the electrical pins 1214.

[0100] Referring now to FIG. 8A, Figure 8 A perspective view of a transparent block 806 formed of solid transparent material 800 is shown for another embodiment of the 1f image array 106B, 608, 708, 708'. The solid transparent material 800 used for the transparent block 806 can be, for example, transparent glass or transparent plastic. A plurality of micro-mirrors 810 in a row and in a serial chain are formed in or on one side of the transparent block 806 of the transparent material 800. A plurality of dichroic or bandpass filters 812 in a row and in a serial chain are formed in or on the opposite side of the transparent block 806 of the transparent material 800. Each dichroic or bandpass filter 812 is tuned to a different wavelength range of light to allow detection of a wide range of fluorescence emitted by a fluorophore. In one embodiment, the plurality of micro-mirrors 810 are concave spherical mirrors.

[0101] The transparent block 806 formed of solid transparent material 800 also includes a 12 degree wedge surface 820 to receive light from a focusing lens as described with reference to the image array 708. Light enters perpendicular to the surface of the wedge surface 820 and is directed (turned) toward the first dichroic or bandpass filter D(l). The light passes through the transparent block 806 to the first dichroic or bandpass filter D(l).

[0102] Referring now to FIG. 8B, Figure 10 A perspective view of a transparent block 1006 formed of solid transparent material 800 is shown for another embodiment of the 1f image array 106B, 608, 708, 708'. The solid transparent material 800 used for the transparent block 1006 can be, for example, transparent glass or transparent plastic. A plurality of micro-mirrors 1010 are concave rectangular mirrors formed into one side of the transparent material. A plurality of dichroic or bandpass filters 1012 are formed in or on the opposite side of the transparent material 800. Each dichroic or bandpass filter 812 is tuned to a different wavelength range of light to allow detection of a wide range of fluorescence emitted by a fluorophore.

[0103] The solid transparent material 800 also includes a 12 degree wedge surface 820 to receive light from a focusing lens as described with reference to the image array 708. Light enters perpendicular to the surface of the wedge surface 820 and is directed (turned) toward the first dichroic or bandpass filter D(l). The light passes through the transparent block to the first dichroic or bandpass filter D(l).

[0104] Referring now to FIG. 8C, Figure 9A A cross-sectional view illustrates the distance (e.g., thickness L) between the spherical micro-mirrors 810 on one side of the transparent block 806 and the opposite side. An axis 814 perpendicular to the transparent block at the center of the spherical micro-mirrors 810 extends to the opposite side of the transparent block 806. Figure 9BAn axis 815 normal to the transparent block is illustrated at the center of the dichroic or bandpass filter 812. The axis 815 extends to the opposite side of the transparent block 806 of the transparent material 800. The axes 814 and 815 are parallel to each other.

[0105] The reflective material 811 is formed (e.g., disposed) on the spherical transparent microlens shape of the solid transparent material 800 to form individual spherical micromirrors 810 on one side of the transparent block 806. The dichroic or bandpass filter 812 is coupled to the material 800 on the opposite side of the transparent block 806.

[0106] Figure 11 Similarly, the distance and axis 1014 between a concave rectangular micromirror 1010 on one side of the transparent material 800 forming the transparent block 1006 and the opposite side are illustrated. Figure 11 A normal axis 1015 at the center point of the dichroic or bandpass filter 1012 is also illustrated. The optical axis 1015 extends to the opposite side of the transparent block 1006 formed by the transparent material 800. The optical axes 1014 and 1015 are parallel to each other.

[0107] Figure 11 The reflective material 1011 is also illustrated as being formed (e.g., disposed) on the curved transparent rectangular shape of the solid transparent block 1006 formed by the transparent material 800 to form the rectangular micromirror 1010. The dichroic or bandpass filter 1012 is coupled to the opposite side of the solid transparent block 1006.

[0108] The fluorescent dyes used in flow cytometry applications span the entire visible and near infrared wavelength range. For long wavelength fluorochromes, the emission wavelength bandwidth is typically larger. Each dichroic or bandpass filter 812 can have its detector filter passband and center wavelength optimized to measure different dyes with the same amount of spectral sampling. Moreover, individual filter optimization allows exclusion of excitation wavelengths from other lasers. In this way, the detector in each channel can be fully utilized to detect the signal of interest. In combination with a fluorescence spectral unmixing algorithm executed by a processor of a computer, individual and optimized passband detection provides for the ultimate detection of a large number of fluorescent dyes of interest.

[0109] Method

[0110] The method of using the various detection systems disclosed herein in a flow cytometer is described below. Fluorescent light of different wavelengths is produced by various fluorochromes excited by a laser, which label different particles in a sample in a flow channel, before the fluorescent light produced by the fluorochromes excited by the laser is emitted out of the end of the optical fiber 102 shown in the figure. The produced fluorescent light is received by a collection lens proximate to the opposite end of the laser, as can be seen in the figure. Figure 13The light fiber 102 couples the fluorescence to the end of the fiber, and thus out of the fiber. The emitted fluorescence has different wavelengths produced by different fluorochromes that have been excited by the laser light attached to different microparticles in the sample fluid.

[0111] The light fiber 102 couples the fluorescence to the end of the fiber, and thus out of the fiber. The emitted fluorescence has different wavelengths produced by different fluorochromes that have been excited by the laser light attached to different microparticles in the sample fluid.

[0112] In the input channel, the light emitted from the end of the fiber is collimated and focused by a lens to a first one of a first plurality of dichroic filters in a first resolving imaging array.

[0113] Further along the input channel, the laser light emitted from the fiber to excite different fluorochromes is blocked by blocking means from interfering with the wavelengths of the detected fluorescence.

[0114] Further along the input channel, the image size from the end of the fiber is magnified to the spot size for the first one of the first plurality of dichroic filters in a series chain or row.

[0115] In the first resolving imaging array, alternatively, the fluorescence of the first wavelength range is reflected between the first plurality of dichroic filters and a first plurality of micromirrors in a series chain or row to collimate the fluorescence on odd numbered dichroic filters and to reimage the fluorescence on even numbered dichroic filters. The focal length of the first plurality of micromirrors and the spacing distance between the first plurality of dichroic filters and the first plurality of micromirrors provide a telescopic effect along the chain of micromirrors to collimate the fluorescence on odd numbered dichroic filters and to reimage the fluorescence on even numbered dichroic filters.

[0116] In the first plurality of dichroic filters in the series chain or row, a bandpass operation is performed on different wavelength ranges of the fluorescence of the first wavelength range at each filter to resolve the wavelength spectrum of the fluorescence of the first wavelength range.

[0117] Adjacent to the first plurality of dichroic filters in the series chain or row is a series chain or row of a plurality of detector channels having a first plurality of first detectors, as shown in Figure 3 , Figure 6 and 7A- Figure 7C Each detector channel has a lens to focus the different wavelength ranges of the fluorescence to a first plurality of light detectors.

[0118] The plurality of detectors of the serial chain or line detect fluorescence in each of the different wavelength ranges in the first wavelength range associated with each of the different fluorochromes tagged to the microparticles. The plurality of light detectors convert each received fluorescence into an electrical signal that can be analyzed and counted.

[0119] As the fluorescence is converted into electrical signals by the detectors, a computer with a processor can then be used to count the number of each different microparticle in the sample fluid, as disclosed in Application No. 15 / 498,397, filed April 26, 2017, entitled COMPACT MULTI-COLOR FLOW CYTOMETER, by David Vrane et al., which is incorporated by reference herein.

[0120] The second and / or third resolving imaging arrays can be used in parallel with the first resolving imaging array. In this case, the method further includes splitting the fluorescence into fluorescence of the first wavelength range for the first resolving imaging array, fluorescence of the second wavelength range for the second resolving imaging array, and / or fluorescence of the third wavelength range for the third resolving imaging array. As Figure 13 As shown, the first optical fiber 102A can be used to direct the fluorescence toward the first resolving imaging array. The second optical fiber 102B can be used to direct the fluorescence toward the second resolving imaging array. The third optical fiber 102B can be used to direct the fluorescence toward the third resolving imaging array.

[0121] The steps described herein for the first resolving imaging array can be performed simultaneously by the second and / or third resolving imaging arrays, so that different additional wavelength ranges can be analyzed. For brevity, repeated steps are not repeated but are incorporated by reference herein.

[0122] These embodiments are thus described. While the embodiments have been particularly described, they should not be construed as limited to such embodiments, but rather construed according to the following claims.

[0123] While particular example embodiments have been described and illustrated, it will be apparent to those skilled in the art that various changes can be made without departing from the spirit and scope of the broad application present herein and that the scope of the present application should not be limited by the specific disclosed embodiments, but should be given the full scope of the appended claims.

[0124] Certain functions of the flow cytometer can be implemented in software and executed by a computer or processor, such as analyzing the electrical signals detected by the detectors to count different microparticles in the sample fluid. The program or code segment using this software performs the tasks necessary to perform these functions. The program or code segment can be stored in a processor-readable medium or transmitted as a computer data signal embodied in a carrier wave over a transmission medium or communication link. The processor-readable medium can include any storage medium that can store information. Examples of processor-readable medium include electronic circuits, semiconductor memory devices, read-only memory (ROM), flash memory, erasable programmable read-only memory (EPROM), floppy disks, CD- ROMs, optical storage, and hard disk. The code segment can be downloaded to the storage medium via a computer network, such as the Internet, an intranet, etc.

[0125] While this specification contains many specifics, these should not be construed as limitations on the scope of the disclosure or of what can be claimed, but rather as descriptions of features specific to particular implementations of the disclosure. Certain features that are described in the context of separate implementations can also be implemented in combination with each other. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations, separately or in sub-combinations. Moreover, although features can be described above as acting in certain combinations and even at times even individually, one or more features from a combination can in some cases be excised from the combination and the combination can be directed to a sub-combination or variation of a sub-combination. Only the claims are limiting at this regard.

Claims

1. A compact wavelength detection module for a flow cytometer, the compact wavelength detection module comprising: a first light detection module that resolves first fluorescence in a first wavelength range, the first light detection module comprising a first plurality of detectors in a first row; a first transparent block adjacent to the first plurality of detectors, a first plurality of micromirrors coupled to a first side of the first transparent block, and a first plurality of filters in a first row coupled to a second side of the first transparent block opposite the first side, each filter of the first plurality of filters reflecting light to one micromirror of the first plurality of micromirrors and passing light of a different wavelength range toward one detector of the first plurality of detectors; and an input channel in optical communication with the first light detection module, wherein the input channel comprises a collimating lens that receives fluorescence from an optical fiber and collimates the fluorescence and a blocking filter that rejects laser light in the fluorescence used to excite the fluorochrome.

2. The compact wavelength detection module for a flow cytometer of claim 1, further comprising: a second light detection module that resolves second fluorescence in a second wavelength range, the second light detection module comprising a second plurality of detectors in a second row; a second transparent block adjacent to the second plurality of detectors, a second plurality of micromirrors coupled to a first side of the second transparent block, and a second plurality of filters in a second row coupled to a second side of the second transparent block opposite the first side, each filter of the second plurality of filters reflecting light to one micromirror of the second plurality of micromirrors and passing light of a different wavelength range toward one detector of the second plurality of detectors. the input channel is in optical communication with the first light detection module and the second light detection module, the input channel receives a beam of fluorescence and splits the beam of fluorescence into the first fluorescence of the first wavelength range and the second fluorescence of the second wavelength range.

3. The compact wavelength detection module for a flow cytometer of claim 2, wherein, 4. The compact wavelength detection module for a flow cytometer of claim 3, wherein the input channel further comprises a wavelength beam splitter that splits a beam of fluorescence into the first fluorescence and the second fluorescence.

5. The compact wavelength detection module for a flow cytometer of claim 4, wherein the input channel further comprises a first focusing lens that focuses the first fluorescence onto a first filter of the first plurality of filters. the first focusing lens is in optical communication with the wavelength beam splitter, and 6. The compact wavelength detection module for a flow cytometer of claim 5, wherein, the input channel further comprises a second focusing lens in optical communication with the wavelength beam splitter that focuses collimated light of the second wavelength range onto a first filter of the second plurality of filters. ​

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