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

Abstract

The invention relates to an optical flow cytometer for fluorescence and scatterometry, comprising:-an incoherent light source intended to generate an illumination beam; -a beam cut-off device comprising at least a first passage allowing passage of a first portion of the illumination beam having a first divergence, the first portion being dedicated to scatter measurement, and a second passage allowing passage of a second portion of the illumination beam having a second divergence, the second portion being dedicated to fluorescence measurement, the second divergence is greater than the first divergence, and the first passage and the second passage are separated by an irradiation beam blocking area of the beam blocking device; -at least one focusing lens for focusing a first portion and a second portion of the illumination beam to a flow chamber comprising an optical interrogation zone; -a flow chamber intended to contain particles flowing through the optical interrogation zone; -a scattering detector for receiving light scattered from a first portion of the illumination beam as particles pass through the optical interrogation zone; -a fluorescence detector for receiving fluorescence emitted by the particles passing through the optical interrogation zone.

Description

[0001] The present invention relates to an optical flow cytometer which characterizes blood cells by performing fluorescence measurement and scattering measurement on a blood cell stream. The present invention relates to the field of automated devices for counting and characterizing particles suspended in a liquid medium, and more particularly to the field of hematology instruments for counting and characterizing various types of cells (such as white blood cells, red blood cells and platelets) and other types of particles (such as algae, bacteria, etc.) contained in a blood sample.

[0002] Therefore, the state of the art and the proposed invention relate to devices for counting and characterizing various types of cells contained in a blood sample 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 measurement and fluorescence measurement.

[0004] Scattering measurement in such an arrangement implies some limitations.

[0005] Unlike fluorescence, single cell scattering is highly anisotropic. Single cell scattering measurement depends not only on the cell parameters to be measured, but also on the illumination beam geometry.

[0006] As the scattering angle increases, the light intensity scattered by a single cell rapidly decays, and it is known that cell parameters that vary greatly need to be reflected according to the angle of the collected scattered light.

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

[0008] In addition, it is also obvious to those skilled in the art that scattered light can only be measured at an angle greater than the divergence of the illumination beam. If the illumination beam has a semi-cone divergence of α (alpha) relative to the optical axis, the scattered light must be collected at an angle not less than α (alpha). Otherwise, a part of the illumination beam will also be collected. As a result, the scattering angle is defined not according to the optical axis, but according to the divergence half-cone of the illumination beam.

[0009] Single cell scattering is highly anisotropic, and the light intensity scattered at a small angle, such as less than 10° (which is measured according to the divergence half-cone of the illumination beam), is quite large. Regardless of the light source, no special sensitivity is required for the photodetector. Standard photodiodes are commonly found in the optical configuration of flow cytometers for detecting small angle scattering. The intensity of the illumination beam is not a key parameter for measuring the small angle scattering of a single blood cell.

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

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

[0012] - the cell parameter (size or complexity) to be measured.

[0013] Fluorescence measurement also implies some limitations.

[0014] Flow cytometers rely to a large extent on fluorescence measurement for the fine characterization of blood cells. Dyes bind to specific cell features (such as its nucleic acid content) and fluoresce when illuminated by light of an appropriate wavelength.

[0015] Known fluorescent dyes have a rather low emission-excitation ratio. Fluorescent emission is usually very weak and requires highly sensitive photodetectors, such as photomultiplier tubes (PMTs), silicon photomultipliers (SiPMs) or avalanche photodiodes (APDs).

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

[0017] Since the fluorescent emission is very weak, it is also advantageous to use a numerical aperture (N.A.) lens (or lens group) to maximize fluorescence collection.

[0018] It is known that fluorescence is isotropic light emission. Similar results will be given regardless of the fluorescence collection direction.

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

[0020] - the highest possible illumination (excitation) intensity, and

[0021] - the maximum possible fluorescence collection.

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

[0023] Unlike a laser, a high-power LED is an extended highly divergent light source. Therefore, optical devices with a high numerical aperture (NA) are required to maximize the light collection efficiency. Since the flow cytometer optical configuration must focus the excitation beam onto the flow chamber (located at the interrogation region), high numerical aperture (NA) focusing optics are also required to handle the highly divergent beam.

[0024] Therefore, the LED limitations are based on a highly divergent illumination (i.e., excitation) beam:

[0025] · a high NA focusing lens, and

[0026] · High-NA scattering collection lens.

[0027] To maximize the illumination (excitation) of the fluorescent dye, high-NA optics must therefore be used for both light collection and focusing of the LED light source. The result of the highly divergent excitation beam passing through the flow cell is that high-NA optics are also required for the measurement of forward scatter, since the scattered light must be measured at a divergence greater than that of the illumination beam.

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

[0029] In an optical flow cytometer that combines fluorescence measurement and scatter measurement, the mechanical design becomes extremely challenging since there is only a small amount of space to mount all the optics along three different walls of the flow cell.

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

[0031] However, regardless of the direction of fluorescence measurement (90°, epi-fluorescence, 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 object of the present invention is to provide a compact optical flow cytometer for performing fluorescence measurement and scatter measurement.

[0033] Another object of the present invention is to eliminate the need for high N.A. forward scatter optics in an optical flow cytometer that combines fluorescence measurement and forward scatter measurement.

[0034] The present invention also aims to ensure the consistency of forward scatter measurement.

[0035] The present invention also aims to solve the mechanical contradictions around the flow cell.

[0036] At least one of the above objects is achieved by an optical flow cytometer for fluorescence measurement and scatter measurement, the optical flow cytometer comprising:

[0037] - An incoherent light source adapted to generate an illumination beam,

[0038] - A beam truncation device comprising at least:

[0039] - A first path that allows a first portion of the illumination beam having a first divergence to pass through, the first portion being dedicated to scatter measurement,

[0040] - A second path that allows a second portion of the illumination beam having a second divergence to pass through, where the second portion is dedicated to fluorescence measurement; the second divergence is greater than the first divergence; the first path and the second path are separated by a region of the beam truncation device that blocks the illumination beam.

[0041] - At least one focusing lens for focusing the first and second portions of the illumination beam onto a flow cell that includes an optical interrogation region.

[0042] - A flow cell that is designed to contain particles flowing through the optical interrogation region.

[0043] - A scattering detector for receiving light scattered from the first portion of the illumination beam when the particles pass through the optical interrogation region.

[0044] - A fluorescence detector for receiving fluorescence emitted by the particles passing through the optical interrogation region.

[0045] The beam truncation device according to the present invention is not a beam splitter. The beam truncation device is used to adjust the cross-section of the illumination beam by truncating a portion of the illumination beam. The input beam cross-section geometry and the output beam cross-section geometry are different. In contrast, with a beam splitter, the input beam and the output beam have the same cross-section geometry.

[0046] The first portion of the illumination beam is dedicated to irradiating the flowing particles to enable precise measurement of forward scattering, especially 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. A gap is created between the first portion and the second portion of the illumination beam according to the present invention. This gap can be advantageously used to collect scattered light.

[0048] With the optical flow cytometer according to the present invention, the scattering measurement is more precise than in the prior art because detection of less than 5° (measured according to the cone half-angle of the illumination beam divergence) is performed without contamination by a second beam that is not set for fluorescence excitation. Precise detection of the forward scattering range defined by [0°, +5°] (measured according to the cone half-angle of the illumination beam divergence) enables precise estimation of the particle size within the sample.

[0049] Since the first and second parts of the illumination beam are angularly separated, it is possible to measure the forward scatter without interference from the second part of the beam dedicated to fluorescence excitation. Thus, forward scatter measurements do not require high numerical aperture (NA) optics. The present invention relieves the mechanical constraints around the flow cell and reduces the cost of the forward scatter acquisition optics. In fact, the optics used for scatter measurement can be smaller than those used in the prior art.

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

[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, an incoherent light source extended at ultraviolet, visible or infrared light wavelengths can be used.

[0053] According to the present invention, the second passage can have a cross-section larger than that of the first passage. Thus, the energy of the second part of the illumination beam is greater than the energy of the first part of the illumination beam.

[0054] The first passage allows the low-divergence part of the illumination beam to pass through, and the second passage allows the high-divergence part of the illumination beam to pass through.

[0055] The divergence is considered to be the maximum tilt angle of the rays constituting the beam. The low-divergence part of the illumination beam is closer to the optical axis than the high-divergence part of the illumination beam.

[0056] The present invention allows decoupling of the high-energy, high-divergence illumination beam required for excitation of the fluorescent dye from the low-energy, low-divergence illumination beam required for forward scatter measurement.

[0057] Since high NA optics are not required, the low-divergence beam is particularly advantageous for forward scatter measurement. Thus, the forward scatter acquisition optics can be moved away 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 present invention, the first passage can be, for example, circular and centered along the optical axis of the illumination beam.

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

[0060] According to an embodiment of the present invention, the second beam truncation device can be arranged relative to the light propagation axis behind the flow cell to block the first part and / or the second part of the illumination beam.

[0061] The second beam truncation device can include a hole 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, in the case where the first passage and the second passage are circular and coaxial, the dimensions can be as follows:

[0064] Radius of the first passage: [0; R]

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

[0066] Radius of the second passage: [n*R; Rmax]

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

[0068] According to the present invention, the beam truncation device can be located on either side of the focusing lens. In the case where the focusing lens is a set of lenses, the beam truncation device can be located within the set of lenses. The beam truncation device does not need to be placed close to the focusing lens. Multiple beam truncation devices can be used at different positions.

[0069] According to the present invention, the beam truncation device can include at least one filter to be placed on the first passage and / or the second passage to change the spectral characteristics of the first part of the illuminating beam and / or the second part of the illuminating 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, the beam truncation device can include at least one polarizer to be placed on the first passage and / or the second passage to change the polarization of the first part of the illuminating beam and / or the second part of the illuminating beam, respectively.

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

[0073] According to the present invention, the beam truncation device can include at least one additional lens to be placed on the first passage and / or the second passage to change the direction of the first part of the illuminating beam and / or the second part of the illuminating beam, respectively.

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

[0075] The filter, polarizer, and direction lens of the beam truncation device can be removable.

[0076] According to a preferred embodiment of the present invention, the beam truncation device can include a diaphragm or consist of a diaphragm, in which the first path and the second path are holes.

[0077] The diaphragm divides through two holes and can be located near the focusing lens of the illuminating beam. These two holes allow for the discrimination of two beams:

[0078] - A first beam with low divergence and low energy for forward scatter measurement, and

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

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

[0081] According to an embodiment of the present invention, the cross-section of the first path and / or the second path can 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 can be centered or not centered with respect to the optical axis.

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

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

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

[0086] According to another embodiment of the present invention, the fluorescence lens is arranged at 90° with respect to the optical axis to collect fluorescence from the flow cell into a fluorescence detector for 90° fluorescence measurement.

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

[0088] According to another embodiment of the present invention, the light collection module after the flow cell can be arranged to detect forward scatter, medium angle scatter, or axial light loss.

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

[0090] When the illumination beam is too divergent, the light scattered at a small angle relative to the illumination beam has a poor correlation with the particle size. Therefore, when the illumination beam is highly divergent, the accuracy of the optical sensor for blood cell characterization deteriorates severely.

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

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

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

[0094] To illustrate the present invention, the currently preferred form is shown in the drawings; however, it should be understood that the present invention is not limited to the specific arrangements and tools described herein.

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

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

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

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

[0099] Figure 5 is a schematic diagram showing the effect of the beam truncation device on the illumination beam;

[0100] Figure 6 is a schematic diagram of low angle scatter detection;

[0101] Figure 7 isFigure 6 Cross-sectional view of the scattered light and the first part of the irradiation beam in

[0102] While the present invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will be described in detail in the present invention. However, it should be understood that the drawings and the detailed description thereof are not intended to limit the present invention to the particular forms disclosed, but on the contrary, the present invention will cover all modifications, equivalents, and substitutions falling within the scope of the present invention defined by the appended claims.

[0103] Hereinafter, the present invention will be described in detail by explaining exemplary embodiments of the present invention with reference to the drawings. The same reference numerals in the drawings denote the same elements.

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

[0105] Referring to Figure 1 , the optical flow cytometer 1 of the present invention includes an irradiation module 2 that generates two irradiation beams 3a and 3b (generated from a single incoherent light source) toward a flow chamber 4, and particles such as blood cells flow in the flow chamber 4. The optical flow cytometer 1 includes an electric device or / and other devices to drive and focus the sample cells or blood cells into a fluid that may or may not be surrounded by sheath fluid. The blood cells circulate through the flow chamber 4.

[0106] The irradiation beams 3a and 3b are focused and guided to vertically pass through the cell stream, and induce fluorescence in the sample particles or their labeled fluorescence. The fluorescence 7 generated by the blood cells is collected by the fluorescence measurement module 5.

[0107] When the blood cells pass through the optical interrogation region, the irradiation beams 3a and 3b also induce scattered light 8. The optical flow cytometer 1 includes a scattered light measurement module 6 that is arranged to collect the scattered light 8 from the flow chamber 4.

[0108] The processor unit 9 is arranged to control the excitation module 2 for the excitation signal. The processor unit 7 also controls the fluorescence measurement module 5 and the scattered light measurement module 6 to detect direct and / or indirect scattered light signals.

[0109] In Figure 1 's embodiment, the scattered light measurement module 6 is arranged on the opposite side of the irradiation module 2, and the fluorescence measurement module 5 is arranged at 90° to the optical axis of the irradiation beam. Other arrangements can be achieved by including mirrors, lenses, and / or beam splitters to deflect the light.

[0110] The invention also relates to an epi-fluorescence configuration (not shown), in which the excitation module 2 may include a fluorescence measurement module 5. In such a configuration, the excitation beam is focused on the flow cell using the same focusing lens, and fluorescence from the flow cell is collected.

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

[0112] The first part 3a is preferably a beam with low energy and low divergence.

[0113] The second part 3b is a beam with high energy and high divergence.

[0114] The two beams 3a and 3b reach the sample in different directions such that the first part 3a and the second part 3b have different directions after passing through the sample.

[0115] According to the invention, there is a gap between the first part 3a and the second part 3b of the illumination beam after passing through the sample. This gap is used to detect scattered light. Since there is no interfering light from the second part 3b of the excitation beam, this detection is considered to be consistent.

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

[0117] Figure 2 Shows the optical details of an optical flow cytometer.

[0118] Module 2 includes an extended incoherent source that emits an illumination beam 3 at 485 nm, such as an LED. The beam truncation device 11 blocks some parts of the excitation beam 3 and includes at least two passages to allow two sub-beams to pass through, which are the first part 3a and the second part 3b of the illumination beam.

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

[0120] The first passage 11A is a circular hole located at the center of the beam truncating device. Thus, the incident axial light passes through this first passage 11a and constitutes the first part 3a of the illuminating beam.

[0121] The second passage 11b has an annular shape concentric with the first passage. The area of the cross-section of the second passage is larger than the area of the cross-section of the first passage. The second part 3b of the illuminating beam includes a beam thicker than the first part 3a, which means higher energy. Since the second passage is centered on the first passage, the second part 3b of the illuminating beam is more divergent than the first part 3a of the excitation beam.

[0122] Figure 4 Another illustration of the beam truncating device is shown, where the passages have different cross-sections. The first passage 11A is an oval hole outside the center of the beam truncating device. The second passage 11b is a square independent of the first passage.

[0123] The beam truncating device according to the present 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 light source).

[0124] On the contrary, the beam truncating device according to the present invention relies on truncating the input beam to change the shape of its cross-section. Those skilled in the art of optics generally know that when using a coherent light source, beam truncation involves strong diffraction patterns. Therefore, the beam truncating device according to the present invention is a segmentation stop that is incompatible with the use of a coherent light source (such as a laser).

[0125] Returning to Figure 2 , the first part 3a of the illuminating beam and the second part 3b of the illuminating beam are focused by the lens 12 on the optical interrogation region 13 in the flow cell 4. The sample is intended to circulate through the optical interrogation region.

[0126] After the flow cell, both the first part 3a and the second part 3b of the illuminating beam are blocked by the second truncating device 14. The device 14 has an annular hole 21 to allow the light 20 scattered by the particles to reach the detector 16.

[0127] Since the second part 3b of the illuminating beam is highly divergent, its angular position is far from the annular hole of the second truncating device 14. Since the scattering intensity decays rapidly with the angle, the part 3b of the illuminating beam only produces a very small scattering intensity through the annular hole of the second truncating device 14.

[0128] On the contrary, the part 3a of the illuminating beam is very close to the annular hole of the second truncating device 14. Therefore, the scattering contribution produced by the part 3a of the illuminating beam through the annular hole of the second truncating device 14 is very high.

[0129] As a result, although the portion 3b of the illumination beam has a much higher power than 3a, since the portion 3b is far from the annular aperture of the second truncation device 14, the portion 3b does not produce a significant scattering contribution through the annular aperture of the second truncation device 14.

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

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

[0132] The first part 3a of the excitation beam and the second part 3b of the excitation beam generate fluorescence through the optical interrogation region 13. The fluorescence lens 17 focuses a part 18 of the fluorescence into the detector 19.

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

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

[0135] Figure 5 The splitting of the illumination beam is shown in detail. The first part 3a of the excitation beam and the second part 3b of the illumination beam are generated from a single light source. The focusing lens causes the first part 3a and the second part 3b to all converge in the flow cell, thereby generating an optical interrogation region.

[0136] After focusing in the sample, the first part 3a remains separated from the second part 3b.

[0137] The proposed innovation eliminates the need for high NA optics for forward scatter measurement. Therefore, it relieves the mechanical constraints around the flow cell and reduces the cost of the forward scatter acquisition optics.

[0138] In the most efficient configuration, the scatter-only beam is a low-divergence central beam. However, the principle of the proposed innovation still applies when the scatter-only beam is not a central beam.

[0139] The present invention allows for improved scatter measurement by detecting scattered light that is not interfered with by the high-intensity fluorescence excitation beam 3b. The quality of the scatter measurement is better, and a small lens can be used to receive the scattered light. Therefore, a large lens with a high numerical aperture for focusing the illumination beam and another large lens with a high numerical aperture for fluorescence detection can be placed around the flow cell.

[0140] The reduction in the size of the lens for collecting scattered light provides space for other lenses used for illumination focusing and fluorescence collection.

[0141] Numerous variations and modifications will be apparent to those skilled in the art upon a full understanding of the above disclosure. It is intended that the appended claims be construed to cover all such variations and modifications.

Claims

1. An optical flow cytometer for fluorescence measurement and scattering measurement, comprising: - An incoherent light source configured to generate an illumination beam, - A beam truncation device including at least: - A first path that allows a first portion of the illumination beam having a first divergence to pass through, and this first portion is dedicated to scattering measurement, - A second path that allows a second portion of the illumination beam having a second divergence to pass through, and this second portion is dedicated to fluorescence measurement; the second divergence is greater than the first divergence; the first path and the second path are separated by a region of the beam truncation device that blocks the illumination beam, - At least one focusing lens for focusing the first portion and the second portion of the illumination beam onto a flow cell including an optical interrogation region, - A flow cell configured to contain particles flowing through the optical interrogation region, - A scattering detector for receiving light scattered from the first portion of the illumination beam when the particles pass through the optical interrogation region, - A fluorescence detector for receiving fluorescence emitted by the particles passing through the optical interrogation region.

2. The optical flow cytometer according to claim 1, characterized in that The incoherent light source is an LED, an incandescent lamp, or an arc lamp.

3. The optical flow cytometer according to claim 1 or 2, characterized in that, The second path has a cross-section larger than that of the first path.

4. The optical flow cytometer according to any one of the preceding claims, characterized in that, The first path is centered along the optical axis of the illumination beam.

5. The optical flow cytometer according to any one of the preceding claims, characterized in that A second beam truncation device 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 according to any one of the preceding claims, characterized in that, When the first path has a circular cross-section with a radius of R, the radial distance between the first path and the second path is greater than or equal to 0.2*R.

7. The optical flow cytometer according to any one of the preceding claims, characterized in that The beam truncation device includes at least one filter to be placed on the first path and / or the second path to respectively change the spectral characteristics of the first portion of the illumination beam and / or the second portion of the illumination beam.

8. The optical flow cytometer according to any one of the preceding claims, characterized in that The beam truncation device includes at least one polarizer to be placed on the first path and / or the second path to respectively change the polarization of the first portion of the illumination beam and / or the second portion of the illumination beam.

9. The optical flow cytometer according to any one of the preceding claims, characterized in that, The beam truncation device includes at least one additional lens to be placed on the first path and / or the second path to respectively change the direction of the first portion of the illumination beam and / or the second portion of the illumination beam.

10. The optical flow cytometer according to any one of the preceding claims, characterized in that, The beam truncation device includes a separator in which the first path and the second path are holes.

11. The optical flow cytometer according to any one of claims 1 to 9, characterized in that, The beam truncation device is a light-shielding material disposed on the lens surface.

12. The optical flow cytometer according to any one of the preceding claims, characterized in that, The cross-section of the first path and / or the second path is circular, square, rectangular, or irregular.

13. The optical flow cytometer according to any one of the preceding claims, characterized in that The focusing lens is designed to collect fluorescence from the flow cell; then, the fluorescence from the flow cell is detected by the fluorescence detector to achieve epi-fluorescence measurement.

14. The optical flow cytometer according to any one of claims 1 to 12, characterized in that, The fluorescence lens is disposed at 90° with respect to the optical axis to converge the fluorescence from the flow cell to the fluorescence detector for 90° fluorescence measurement.

15. The optical flow cytometer according to any one of the preceding claims, characterized in that, The scattering detector is arranged to detect light scattered by flowing particles at an angle within the range of [0°, 5°] measured according to the divergence of the first portion of the illumination beam.

16. The optical flow cytometer according to any one of the preceding claims, characterized in that A detection lens or a set of lenses is provided after the flow cell to collect the light scattered by the flowing particles and focus the light scattered by the flowing particles onto the scatter detector.

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