Optical flow cytometer for fluorescence and scatter measurements by splitting a light beam emitted by a single incoherent light source

Abstract

The invention relates to an optical flow cytometer for fluorescence and scatter measurements, comprising: - a non-coherent light source intended to generate an illumination beam; - a beam truncation device comprising at least a first passage allowing a first portion of the illumination beam having a first divergence to pass, the first portion being dedicated to scatter measurements, and a second passage allowing a second portion of the illumination beam having a second divergence to pass, the second portion being dedicated to fluorescence measurements, the second divergence being greater than the first divergence, the first passage and the second passage being separated by a region of the beam truncation device blocking the illumination beam; - at least one focusing lens for focusing the first portion and the second portion of the illumination beam to a flow cell comprising an optical interrogation zone; - a flow cell intended to contain particles flowing through the optical interrogation zone; - a scatter detector for receiving light scattered from the first portion of the illumination beam when a particle passes through the optical interrogation zone; - a fluorescence detector for receiving fluorescence emitted by a particle passing through the optical interrogation zone.

Description

[0001] This invention relates to optical flow cytometers, which characterize blood cells by measuring fluorescence and scattering of blood cell flows. This invention also relates to the field of automated apparatus for counting and characterizing particles suspended in liquid media, and particularly to the field of hematological instruments for counting and characterizing various types of cells (e.g., white blood cells, red blood cells, and platelets) and other types of particles (e.g., algae, bacteria, etc.) contained in blood samples.

[0002] Therefore, the current state of the technology and the proposed invention relate to apparatus for counting and characterizing various types of cells contained in blood samples and for determining their relative distribution in various subpopulations based on light scattering and fluorescence.

[0003] It is known that LED light sources can be used in optical flow cytometers for scattering and fluorescence measurements.

[0004] Scattering measurements in this arrangement imply some limitations.

[0005] Unlike fluorescence, single-cell scattering is highly anisotropic. Single-cell scattering measurements depend not only on the cellular parameters being measured but also on the geometry of the illumination beam.

[0006] As the scattering angle increases, the intensity of light scattered by a single cell decreases rapidly, and it is known that cell parameters vary greatly depending on the angle of the collected scattered light.

[0007] Those skilled in the art will know that light scattered at a small angle by a single blood cell reflects the size of that single blood cell. Light scattered at the correct angle, however, depends on the internal complexity of the blood cell (number of nuclear lobes, granularity, etc.).

[0008] Furthermore, it will be equally apparent to those skilled in the art that scattered light can only be measured at angles exceeding the divergence of the illuminating beam. If the illuminating beam has a semi-conical divergence of α (alpha) relative to the optical axis, then the scattered light must be collected from an angle not less than α (alpha). Otherwise, a portion of the illuminating beam will also be collected. As a result, the scattering angle is not defined according to the optical axis, but rather according to the divergence half-cone of the illuminating beam.

[0009] Individual cell scattering is highly anisotropic, and the intensity of light scattered at small angles, such as less than 10° (measured from the half-angle of the cone of divergence of the illumination beam), is quite considerable. Regardless of the light source, no special sensitivity is required for the photodetector. Standard photodiodes are commonly found in the optical configurations of flow cytometers and are used to detect small-angle scattering. The intensity of the illumination beam is not a key parameter for measuring small-angle scattering from individual blood cells.

[0010] Therefore, the direction of light collection from a single cell depends on:

[0011] - The divergence of the illumination beam, and

[0012] - Cell parameters to be measured (size or complexity).

[0013] Fluorescence measurements also imply some limitations.

[0014] Flow cytometry relies heavily on fluorescence measurements for fine characterization of blood cells. Dyes bind to specific cellular features (such as their nucleic acid contents) and fluoresce when illuminated with light of an appropriate wavelength.

[0015] Fluorescent dyes are known to have a relatively low emission-excitation ratio. Fluorescence emission is typically very weak and requires highly sensitive photodetectors, such as photomultiplier tubes (PMTs), silicon photomultiplier tubes (SiPMs), or avalanche photodiodes (APDs).

[0016] The intensity of fluorescence emission is also proportional to the intensity of dye excitation (or irradiation). Therefore, it is advantageous to have the highest possible intensity of excitation (or irradiation) so that fluorescence emission is also maximized.

[0017] Since fluorescence emission is very weak, it is also advantageous to use a numerical aperture (NA) lens (or lens group) to maximize fluorescence acquisition.

[0018] Fluorescence is known to be isotropic emission. Regardless of the direction of fluorescence collection, similar results will be given.

[0019] Therefore, the limitations of fluorescence measurement are as follows:

[0020] -The highest possible irradiation (excitation) intensity, and

[0021] -Maximize the possible fluorescence acquisition.

[0022] Using extended incoherent light sources also implies some limitations.

[0023] Unlike lasers, high-power LEDs are extended, highly divergent light sources. Therefore, high numerical aperture (NA) optics are required to maximize light collection efficiency. Since flow cytometer optics must focus the excitation beam into the flow cell (located in the interrogation zone), high numerical aperture (NA) focusing optics are also needed to handle the highly divergent beam.

[0024] Therefore, the LED confinement is based on a high-divergence illumination (i.e., excitation) beam:

[0025] High-NA focusing lenses, and

[0026] • High-NA scattering acquisition lens.

[0027] To maximize the irradiation (excitation) of the fluorescent dye, high-NA optics must be used for both light collection and focusing of the LED light source. The highly divergent excitation beam passing through the flow cell results in the need to measure the scattered light at a level exceeding the divergence of the irradiation beam; therefore, high-NA optics are also required for measuring forward scattering.

[0028] Since fluorescence measurements also require high-NA optics, all optical functions (light source focusing, forward scattering (FSC) measurement, and fluorescence measurement) require high-NA optics.

[0029] In optical flow cytometers that combine fluorescence and scattering measurements, the mechanical design becomes extremely challenging because there is only a small space available to fix all the optics along the three different walls of the flow chamber.

[0030] As prior art, document WO2019 / 058152 discloses an epifluorescence optical flow cytometer, in which the same lens is used for focusing the excitation beam and for detecting fluorescence.

[0031] However, regardless of the direction of fluorescence measurement (90°, epifluo, any other direction), a high-NA lens is still required to collect the fluorescence. This results in strong mechanical constraints around the flow cell: there is not enough space for multiple high-NA optics.

[0032] The purpose of this invention is to provide a compact optical flow cytometer for performing fluorescence and scattering measurements.

[0033] Another object of the present invention is to eliminate the need for high-NA forward scattering optics in optical flow cytometers that combine fluorescence measurement and forward scattering measurement.

[0034] This invention also aims to ensure the consistency of forward scattering measurements.

[0035] The present invention also aims to resolve the mechanical contradictions surrounding the flow chamber.

[0036] At least one of the above objectives is achieved by an optical flow cytometer for fluorescence and scattering measurements, the optical flow cytometer comprising:

[0037] -A non-coherent light source designed to produce an illuminating beam.

[0038] - Beam interruption device, comprising at least:

[0039] - A first path, which allows a first portion of the illumination beam having a first divergence to pass through, the first portion being dedicated to scattering measurements.

[0040] - A second path, which allows a second portion of the illumination beam with a second divergence to pass through, the second portion being dedicated to fluorescence measurement; the second divergence is greater than the first divergence; the first and second paths are separated by the region of the illumination beam blocked by the beam-cutting device.

[0041] - At least one focusing lens for focusing a first portion and a second portion of the illumination beam onto a flow chamber including an optical interrogation area.

[0042] - A flow chamber designed to contain particles flowing through the optical interrogation zone.

[0043] - A scattering detector, which is used to receive light scattered from a first portion of the illuminating beam as a particle passes through the optical interrogation zone.

[0044] - A fluorescence detector, which is used to receive fluorescence emitted by particles passing through the optical interrogation zone.

[0045] The beam truncation device according to the invention is not a beam splitter. A beam truncation device is used to adjust the cross-section of an illuminating beam by partially cutting off the beam. The cross-sectional geometry of the input beam and the cross-sectional geometry of the output beam are different. Conversely, with a beam splitter, the input beam and the output beam have the same cross-sectional geometry.

[0046] The first part of the illumination beam is dedicated to irradiating the flowing particles, enabling precise measurement, especially of forward scattering around the optical axis.

[0047] The second portion of the illumination beam is dedicated to irradiating the flowing particles via one or more directions different from the direction of the first portion of the illumination beam. According to the invention, a gap is created between the first and second portions of the illumination beam. This gap can be advantageously used to collect scattered light.

[0048] With the optical flow cytometer according to the invention, scattering measurements are more accurate than in the prior art because detection of less than 5° (measured according to the half-angle of the cone of the irradiation beam divergence) is performed without contamination from a second beam used for fluorescence excitation. Precise detection of the forward scattering range defined by [0°, +5°] (measured according to the half-angle of the cone of the irradiation beam divergence) enables accurate estimation of particle size within the sample.

[0049] Because the first and second portions of the illumination beam are angularly separated, forward scattering can be measured without interference from the second portion of the beam dedicated to fluorescence excitation. Therefore, forward scattering measurements do not require high numerical aperture (NA) optics. This invention removes the mechanical constraints surrounding the flow cell and reduces the cost of forward scattering acquisition optics. In fact, the optics used for scattering measurements can be smaller than those used in the prior art.

[0050] According to the present invention, scattering measurements can be performed simultaneously with fluorescence measurements.

[0051] According to the present invention, the incoherent light source can be an LED, an incandescent lamp, or an arc lamp.

[0052] Different light sources with different wavelengths can be used. For example, incoherent light sources with extended wavelengths of ultraviolet, visible, or infrared light can be used.

[0053] According to the present invention, the second path can have a larger cross-section than the first path. Therefore, the energy of the second portion of the irradiating beam is greater than the energy of the first portion of the irradiating beam.

[0054] The first path allows the low-divergence portion of the illumination beam to pass through, while the second path allows the high-divergence portion of the illumination beam to pass through.

[0055] Divergence is considered to be the maximum tilt angle of the rays that make up a beam. The low-divergence portion of an illuminating beam is closer to the optical axis than the high-divergence portion.

[0056] This invention allows for the decoupling of the high-energy, high-divergence irradiation beam required for the excitation of fluorescent dyes from the low-energy, low-divergence irradiation beam required for forward scattering measurements.

[0057] Because high-NA optics are not required, low-divergence beams are particularly advantageous for forward scattering measurements. Therefore, the forward scattering acquisition optics can be removed from the flow cell, and low-NA optics can be used, thereby reducing the cost of the optics.

[0058] According to a preferred embodiment of the invention, the first path may be, for example, circular and centered along the optical axis of the irradiating beam.

[0059] The second pathway can be circular and coaxial with the first pathway.

[0060] According to an embodiment of the present invention, the second beam cutting device may be disposed after the flow chamber relative to the optical propagation axis to block the first and / or second portions of the irradiating beam.

[0061] The second beam-cutting device may include an aperture that allows only all or part of the scattered light to pass through.

[0062] According to a preferred embodiment of the present invention, when the first passage has a circular cross-section with a radius of R, the radial distance between the first passage and the second passage can be greater than or equal to 0.2*R.

[0063] For example, if the first and second channels are circular and coaxial, the dimensions can be as follows:

[0064] First path radius: [0; R]

[0065] Blocking radius: [R; n*R], where n>1.2

[0066] Second path radius: [n*R; Rmax]

[0067] Preferably, the cross-section of the first path is several times smaller than that of the second path to generate a high-power, highly divergent beam for fluorescence measurements and another low-energy, low-divergence beam for consistent forward scattering measurements.

[0068] According to the present invention, the beam cutting device can be located on either side of the focusing lens. If the focusing lens is a set of lenses, the beam cutting device can be located within that set of lenses. The beam cutting device does not need to be placed close to the focusing lens. Multiple beam cutting devices can be used in different locations.

[0069] According to the present invention, the beam cutting device may include at least one filter to be placed on the first path and / or the second path to change the spectral characteristics of the first portion and / or the second portion of the irradiated beam, respectively.

[0070] The filter type can be colored glass (e.g., Schott BG12) or an interference filter (e.g., Semrock 447 / 60nm BrightLine).

[0071] According to the present invention, a beam truncation device may include at least one polarizer to be placed on a first path and / or a second path to change the polarization of a first portion and / or a second portion of the irradiated beam, respectively.

[0072] The polarizer can be linear (e.g., Edmund Optics linear polarizing film, number: 19-003), circular (e.g., Edmund Optics circular polarizer, number: 88-095), or waveplate (e.g., Edmund Optics number: 91-012).

[0073] According to the present invention, the beam cutting device may include at least one additional lens to be placed on the first path and / or the second path to change the direction of the first portion and / or the second portion of the irradiated beam, respectively.

[0074] For example, a small plano-convex lens (e.g., Edmund Optics number: 49-173) can be placed in the first path.

[0075] The filters, polarizers, and direction lenses of the beam cutoff device can be removable.

[0076] According to a preferred embodiment of the present invention, the beam cutting device may include or be composed of a diaphragm, wherein the first passage and the second passage are holes.

[0077] The separator achieves separation through two holes and can be located near the focusing lens that illuminates the beam. These two holes allow for the differentiation of the two beams:

[0078] - A low-divergence, low-energy first beam for forward scattering measurements, and

[0079] - A high-divergence, high-energy second beam used for the excitation of fluorescent dyes.

[0080] According to an embodiment of the present invention, the first beam cutting device and / or the second beam cutting device may be a light-shielding material disposed on the lens surface.

[0081] According to embodiments of the present invention, the cross-section of the first passage and / or the second passage may be circular, square, rectangular or irregular.

[0082] According to an embodiment of the present invention, the cross-section of the first path and / or the second path may be centered or may not be centered relative to the optical axis.

[0083] According to the present invention, the focusing lens may be a single lens or a group of lenses.

[0084] According to an embodiment of the present invention, the focusing lens may also be designed to collect fluorescence from the flow cell; then, the fluorescence from the flow cell is detected by a fluorescence detector to achieve epifluorescence measurement.

[0085] In this embodiment, the focusing lens is used to focus the illumination beam onto the sample and to collect fluorescence from the sample.

[0086] According to another embodiment of the invention, the fluorescent lens is set at 90° relative to the optical axis to collect fluorescence from the flow cell to the fluorescence detector for 90° fluorescence measurement.

[0087] The present invention can advantageously include 90-degree fluorescence measurement or epifluorescence. In both cases, the present invention ensures consistent forward scattering measurement.

[0088] According to another embodiment of the invention, the light acquisition module after the flow chamber can be arranged to detect forward scattering, medium-angle scattering, or axial light loss.

[0089] According to a preferred embodiment of the invention, the scattering detector can be arranged to detect light scattered by flowing particles at angles within the range of [0°, 5°] (this angle range is measured based on the divergence of a first portion of the irradiating beam). Other angle ranges, such as [0°, 10°] or [0°, 15°], can be used.

[0090] When the illumination beam is too divergent, the correlation between light scattered at small angles relative to the illumination beam and particle size is poor. Therefore, when the illumination beam is highly divergent, the accuracy of the optical sensor in characterizing blood cells is severely degraded.

[0091] This invention is based on beam splitting and blocking, which generates a high-power, highly divergent beam for fluorescence measurements and another low-energy, low-divergence beam for consistent forward scattering measurements.

[0092] According to an embodiment of the present invention, a detection lens or a set of lenses may be provided after the flow chamber to collect light scattered by the flowing particles and focus the light scattered by the flowing particles onto the scattering detector.

[0093] The scattering detector can be placed directly on or near the optical interrogation area to directly detect scattered light.

[0094] For the purpose of illustrating the invention, the accompanying drawings show the currently preferred form; however, it should be understood that the invention is not limited to the arrangements and tools explicitly described herein.

[0095] Figure 1 This is an overall view showing the components of an optical flow cytometer according to the present invention;

[0096] Figure 2 This is a schematic diagram illustrating the arrangement of optical components and light trajectories in an example of an optical flow cytometer according to the present invention;

[0097] Figure 3 This is a schematic front view of the beam-cutting device according to the present invention;

[0098] Figure 4 This is a schematic front view of another beam-cutting device according to the present invention, and

[0099] Figure 5 This is a schematic diagram illustrating the effect of the beam-cutting device on the irradiating beam;

[0100] Figure 6 This is a schematic diagram of low-angle scattering detection;

[0101] Figure 7 yes Figure 6 A cross-sectional view of the first part of the scattered light and the illuminating beam.

[0102] While the invention is readily adaptable to various modifications and alternatives, specific embodiments thereof are illustrated by way of example in the accompanying drawings and will be described in detail herein. However, it should be understood that the drawings and their detailed description are not intended to limit the invention to the specific forms disclosed; rather, the invention is intended to cover all modifications, equivalents, and substitutions falling within the scope of the invention as defined by the appended claims.

[0103] The invention will now be described in detail with reference to the accompanying drawings, which illustrate exemplary embodiments of the invention. Like reference numerals in the drawings denote like elements.

[0104] Figure 1 This is an overall view illustrating the components of an optical flow cytometer according to an exemplary embodiment of the present invention.

[0105] refer to Figure 1 The optical flow cytometer 1 of the present invention includes an illumination module 2 for generating two illumination beams 3a and 3b (generated from a single incoherent light source) toward a flow chamber 4, in which particles such as blood cells flow. The optical flow cytometer 1 includes motorized and / or other means to drive and focus sample cells or blood cells into a fluid surrounded or not surrounded by sheath fluid. Blood cells circulate through the flow chamber 4.

[0106] Irradiation beams 3a and 3b are focused and guided vertically through the cell stream, inducing fluorescence in the fluorescent sample particles or their labels. The fluorescence 7 generated by the blood cells is acquired by the fluorescence measurement module 5.

[0107] As blood cells pass through the optical interrogation zone, the illumination beams 3a and 3b also induce scattered light 8. The optical flow cytometer 1 includes a scattering measurement module 6, which is configured to collect the scattered light 8 from the flow chamber 4.

[0108] Processor unit 9 is configured to control excitation module 2 for excitation signal. Processor unit 7 also controls fluorescence measurement module 5 and scattering measurement module 6 to detect direct and / or indirect scattering signals.

[0109] exist Figure 1 In this embodiment, the scattering measurement module 6 is arranged on the opposite side of the illumination module 2, and the fluorescence measurement module 5 is arranged at 90° to the optical axis of the illumination beam. Other arrangements can be made, including mirrors, lenses, and / or beam splitters, to deflect the light.

[0110] The present invention also relates to an epifluorescence configuration (not shown), in which the excitation module 2 may include a fluorescence measurement module 5. In this configuration, the same focusing lens is used to focus the excitation beam onto the flow cell and to collect fluorescence from the flow cell.

[0111] According to the present invention, the irradiation beam is divided into a first part 3a of the excitation beam and a second part 3b of the excitation beam.

[0112] Part 3a is preferably a beam with low energy and low divergence.

[0113] Part 2, 3b, is a high-energy beam with high divergence.

[0114] Two beams, 3a and 3b, arrive at the sample in different directions, causing the first part 3a and the second part 3b to present different directions after passing through the sample.

[0115] According to the present invention, after the sample passes through, a gap exists between the first portion 3a and the second portion 3b of the irradiating beam. This gap is used to detect scattered light. Since there is no interfering light exciting the second portion 3b of the beam, the detection is considered consistent.

[0116] For example, scattering measurements within the range of [0°, 5°] (based on the cone half-angle of the beam divergence) can be performed in this gap. In practice, 5° is considered to be outside the cross-section of the first portion 3a of the beam. Figure 6 The 5-degree detection is shown in the gap of the excitation beam without the second part 3b. The scattering measurement is therefore more accurate. Figure 7 yes Figure 6 A cross-sectional view of the first part of the scattered light and the illuminating beam. The first part 3a of the illuminating beam is a circle surrounded by the crown representing the scattered light at 5°.

[0117] Figure 2 The optical details of the optical flow cytometer are shown.

[0118] Module 2 includes an extended incoherent source, such as an LED, that emits a 485nm illumination beam 3. The beam cutting device 11 blocks portions of the excitation beam 3 and includes at least two pathways to allow two sub-beams to pass through, namely a first portion 3a and a second portion 3b of the illumination beam.

[0119] Figure 3 An example of a beam-cutting device according to the invention is shown. It includes two passages, apertures, or transparent materials (at the excitation wavelength) 11a and 11b to allow a first portion 3a and a second portion 3b of the excitation beam to pass through, respectively. Other rays of the excitation beam are blocked by an opaque material 11c.

[0120] The first passage 11A is a circular hole located at the center of the beam cutting device. Therefore, the irradiated axial light passes through the first passage 11A and forms the first part 3a of the irradiated beam.

[0121] The second path 11b has a ring shape concentric with the first path. The cross-sectional area of ​​the second path is larger than that of the first path. The second portion 3b of the irradiating beam includes a thicker beam than the first portion 3a, which means higher energy. Since the second path is centered on the first path, the second portion 3b of the irradiating beam is more divergent than the first portion 3a of the excitation beam.

[0122] Figure 4 Another illustration of the beam-cutting device is shown, wherein the passages have different cross-sections. The first passage 11A is an elliptical aperture outside the center of the beam-cutting device. The second passage 11b is square, independent of the first passage.

[0123] The beam truncation device according to the invention is not a beam splitter. A standard beam splitter does not involve any kind of truncation of the input beam and can be used with any kind of light source (coherent or incoherent).

[0124] Conversely, the beam truncation device according to the invention relies on truncation of the input beam to change the shape of its cross-section. Those skilled in the art of optics generally know that beam truncation involves strong diffraction patterns when using coherent light sources. Therefore, the beam truncation device according to the invention is a segmentation stop incompatible with the use of coherent light sources (e.g., lasers).

[0125] Back Figure 2 The first portion 3a and the second portion 3b of the illumination beam are focused by lens 12 onto the optical interrogation zone 13 within the flow chamber 4. The sample is intended to circulate through the optical interrogation zone.

[0126] After the flow chamber, both the first portion 3a and the second portion 3b of the illumination beam are blocked by the second interception device 14. The device 14 has an annular aperture 21 to allow the light 20 scattered by the particles to reach the detector 16.

[0127] Since the second portion 3b of the irradiating beam is highly divergent, its angular position is far from the annular aperture of the second cutoff device 14. Because the scattering intensity decays rapidly with angle, the portion 3b of the irradiating beam only produces a very small scattering intensity through the annular aperture of the second cutoff device 14.

[0128] Conversely, portion 3a of the irradiating beam is very close to the annular aperture of the second cutoff device 14. Therefore, the scattering contribution of portion 3a of the irradiating beam through the annular aperture of the second cutoff device 14 is very high.

[0129] As a result, although the portion 3b of the irradiated beam has much higher power than 3a, it does not contribute significantly to scattering through the annular aperture of the second cutoff device 14 because it is far from the annular aperture.

[0130] The detection lens 15 is used to guide the light scattered by the particles to the photodetector 16.

[0131] Lens 15 and photodetector 16 are Figure 1 The components of the scattering measurement module 6.

[0132] The first portion 3a and the second portion 3b of the excitation beam generate fluorescence through the path of the optical interrogation zone 13. The fluorescent lens 17 focuses a portion 18 of this fluorescence onto the detector 19.

[0133] Fluorescent lens 17 and detector 19 are Figure 1 The components of the fluorescence measurement module 5.

[0134] With the arrangement of the present invention, the detection lens 15 can be small, which means that there are fewer mechanical constraints on mounting the large (high numerical aperture) focusing lens 12 and the fluorescence lens 17.

[0135] Figure 5 The segmentation of the illumination beam is shown in detail. The first portion 3a of the excitation beam and the second portion 3b of the illumination beam are generated from a single light source. A focusing lens causes the first portion 3a and the second portion 3b to converge entirely within the flow chamber, thereby creating the optical interrogation zone.

[0136] After focusing on the sample, the first part 3a and the second part 3b remain separated.

[0137] The proposed innovation eliminates the need for high-NA optics for forward scattering measurements. This removes the mechanical constraints surrounding the flow cell and reduces the cost of forward scattering acquisition optics.

[0138] In the most efficient configuration, the scattering-specific beam is a low-divergence center beam. However, the innovative principle proposed still applies when the scattering-specific beam is not a center beam.

[0139] This invention allows for improved scattering measurements by detecting scattered light that is not interfered with by the high-intensity fluorescence excitation beam 3b. The quality of the scattering measurement is better, and the scattered light can be received using small lenses. Therefore, large lenses with high numerical apertures can be placed around the flow chamber for focusing the illumination beam, and another large lens with a high numerical aperture can be used for fluorescence detection.

[0140] The reduction in the size of the lens used to collect scattered light has made room for other lenses used for illumination focusing and fluorescence collection.

[0141] Upon fully understanding the foregoing disclosure, numerous variations and modifications will be apparent to those skilled in the art. The appended claims are intended to be construed as encompassing all such variations and modifications.

Claims

1. An optical flow cytometer for fluorescence and scatter measurements, comprising: - an incoherent light source (10) intended to produce an illumination beam (3), - a beam truncation device (11) comprising at least: - a first passage (11a) allowing a first portion (3a) of the illumination beam (3) having a first divergence to pass, the first portion (3a) being dedicated to scatter measurements, - a second passage (11b) allowing a second portion (3b) of the illumination beam (3) having a second divergence to pass, the second portion (3b) being dedicated to fluorescence measurements; the second divergence being greater than the first divergence; the first passage and the second passage being separated by a region of the beam truncation device blocking the illumination beam, - at least one focusing lens (12) for focusing the first portion (3a) and the second portion (3b) of the illumination beam to a flow cell (4) comprising an optical interrogation zone (13), - a flow cell (4) intended to contain particles flowing through the optical interrogation zone; the beam truncation device (11) and the at least one focusing lens (12) being optically located between the light source and the flow cell (4), - a scatter detector (16) for receiving light (20) scattered from the first portion (3a) of the illumination beam when a particle crosses the optical interrogation zone, - a fluorescence detector (19) for receiving fluorescence (18) emitted by a particle crossing the optical interrogation zone.

2. The optical flow cytometer of claim 1, wherein, The incoherent light source (10) is a LED, an incandescent lamp or an arc lamp.

3. The optical flow cytometer of claim 1 or 2, wherein, The second passage (11b) has a cross section greater than the cross section of the first passage (11a).

4. The optical flow cytometer of claim 1 or 2, wherein, The first passage (11a) is centered along the optical axis of the illumination beam.

5. The optical flow cytometer of claim 1 or 2, wherein, A second beam truncation device (14) is provided after the flow cell to block the first portion and / or the second portion of the illumination beam.

6. The optical flow cytometer of claim 1 or 2, wherein, When the first passage (11a) has a circular cross section with a radius R, the radial distance between the first passage and the second passage is greater than or equal to 0.2*R.

7. The optical flow cytometer of claim 1 or 2, wherein, The beam truncation device (11) comprises at least one optical filter to be placed on the first passage (11a) and / or on the second passage (11b) to respectively modify the spectral properties of the first portion of the illumination beam and / or of the second portion of the illumination beam.

8. The optical flow cytometer of claim 1 or 2, wherein, The beam truncation device (11) comprises at least one polarizer to be placed on the first passage (11a) and / or on the second passage (11b) to respectively modify the polarization of the first portion of the illumination beam and / or of the second portion of the illumination beam.

9. The optical flow cytometer of claim 1 or 2, wherein, The beam truncation device (11) comprises at least one additional lens to be placed on the first passage (11a) and / or on the second passage (11b) to respectively modify the direction of the first portion of the illumination beam and / or of the second portion of the illumination beam.

10. The optical flow cytometer of claim 1 or 2, wherein, The beam truncation device comprises a partition in which the first passage and the second passage are holes.

11. The optical flow cytometer of claim 1 or 2, wherein, The light beam truncation device (11) is a light blocking material disposed on the lens surface.

12. The optical flow cytometer of claim 1 or 2, wherein, The first passage (11a) and / or the second passage (11b) is circular, square, rectangular or irregular in cross-section.

13. The optical flow cytometer of claim 1 or 2, wherein, The focusing lens (12) is designed to collect fluorescence from the flow cell (4); then, the fluorescence from the flow cell is detected by the fluorescence detector to achieve epi-fluorescence measurement.

14. The optical flow cytometer of claim 4, wherein, The fluorescence lens (12) is disposed at 90° relative to the optical axis to converge fluorescence from the flow cell to the fluorescence detector for 90° fluorescence measurement.

15. The optical flow cytometer of claim 1 or 2, wherein, The scatter detector (16) is arranged to detect light scattered by the flowing particles at an angle in the range of [0°, 5°] measured from the divergence of the first portion of the illumination light beam.

16. The optical flow cytometer of claim 1 or 2, wherein, A detection lens (15) or a set of lenses is disposed after the flow cell to collect light scattered by the flowing particles and focus the light scattered by the flowing particles to the scatter detector.

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