An optical flow cytometer that measures fluorescence and scatter by splitting the beam emitted from a single incoherent light source

Abstract

The present 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, the first path for passing a first portion of the illumination beam at a first divergence angle, the first portion being for scatter measurements; a second path for passing a second portion of the illumination beam at a second divergence angle, the second portion being for fluorescence measurements, the second divergence angle being greater than the first divergence angle; and the first and second paths being arranged such that: The device comprises a beam truncation device having at least a second passage separated by a region of the beam truncation device that blocks the illumination beam, at least one focusing lens that focuses the first and second portions of the illumination beam onto a flow cell including an optical inspection zone, the flow cell intended to contain particles flowing through the optical inspection zone, a scattering detector that receives light scattered from the first portion of the illumination beam as the particles traverse the optical inspection zone, and a fluorescence detector that receives fluorescence emitted by particles traversing the optical inspection zone.

Description

[Technical Field]

[0001] The present invention relates to an optical flow cytometer for characterizing blood cells by making fluorescence and scatter measurements on a flow of blood cells. 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, as well as other types of particles, such as algae and bacteria, contained in a blood sample.

[0002] Therefore, the prior art and the proposed invention relate to an apparatus for counting and characterizing different cell types contained in a blood sample and determining their relative distribution within different subpopulations based on light scattering and fluorescence. [Background technology]

[0003] In optical flow cytometers, it is known to use LED light sources to perform scatter and fluorescence measurements.

[0004] Scattering measurements in such a configuration have several limitations.

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

[0006] It is also known that the light intensity scattered by a single cell decays rapidly with increasing scattering angle and reflects significantly different cellular parameters depending on the angle of the focused scattered light beam.

[0007] Those skilled in the art will recognize that the small angle scattered light from a single blood cell reflects the size of that cell, while the right angle scattered light depends on the internal complexity of the blood cell (number of nuclear lobes, granularity, etc.).

[0008] Furthermore, it will be apparent to those skilled in the art that scattered light can only be measured above the divergence angle of the illumination beam. If the divergence angle of the half-cone of the illumination beam is α (alpha) relative to the optical axis, then scattered light must be collected from angles equal to or greater than α (alpha), otherwise part of the illumination beam will also be collected. Thus, the scattering angle is determined by the half-cone of the divergence angle of the illumination beam, not the optical axis.

[0009] Scattering from single cells is highly anisotropic, and the light intensity of small-angle scattering, for example, less than 10 degrees (measured from the half-cone of divergence of the illumination beam), is very strong. Regardless of the light source, no special sensitivity is required from the photodetector. Standard photodiodes are common in flow cytometry optics for measuring small-angle scattering. The intensity of the illumination beam is not a critical parameter for measuring small-angle scattering from single blood cells.

[0010] Therefore, the direction to collect the scattered light from a single cell is - the divergence angle of the irradiating beam, and - Cell parameters to be measured (size or complexity) It depends on.

[0011] Fluorescence measurements also have some limitations.

[0012] Flow cytometry can rely heavily on fluorescence measurements to characterize blood cells in detail: dyes bind to specific cellular properties (such as nucleic acid content) and emit fluorescence when illuminated with the appropriate wavelength.

[0013] Fluorescent dyes are known to have a relatively low emission-to-excitation ratio, and their fluorescence emission is usually very weak, requiring highly sensitive photodetectors such as photomultiplier tubes (PMTs), silicon photomultiplier tubes (SiPMs), or avalanche photodiodes (APDs).

[0014] The intensity of the fluorescent emission is also proportional to the intensity of the excitation (or illumination) of the dye, so it is advantageous to make the intensity of the excitation (or illumination) as high as possible so that the fluorescent emission is also maximized.

[0015] Because the emission from fluorescence is very weak, it is also advantageous to use a high numerical aperture (NA) lens (or lens group) to maximize collection of the fluorescence.

[0016] Fluorescence is known to be an isotropic emission, so whatever the direction of collection of the fluorescence, the results will be almost the same.

[0017] Therefore, the constraints of fluorescence measurements are as follows: - the highest possible illumination (excitation) intensity, and - Collect the widest possible range of fluorescence.

[0018] The use of a non-coherent extended light source also comes with some limitations.

[0019] Unlike lasers, high-power LEDs are highly divergent, extended light sources, which requires high numerical aperture (NA) optics to maximize light collection efficiency. Because flow cytometry optics must focus the excitation beam onto the flow cell (test area), high numerical aperture (NA) collection optics are also required to handle the highly divergent beam.

[0020] Therefore, the limitations of LEDs are based on a highly divergent illumination (i.e., excitation) beam: High NA focusing lens, and High NA scatter collecting lens.

[0021] To maximize illumination (excitation) of the fluorophores, it is essential to use high NA optics to both collect and focus the light from the LED light source. Because the highly divergent excitation beam passes through the flowcell, high NA optics are also required to measure forward scatter, since scattering must be measured beyond the divergence angle of the illumination beam.

[0022] High NA optics are also required for fluorescence measurements, so high NA optics are required for all optical functions (light source focusing, forward scatter (FSC) measurement, fluorescence measurement).

[0023] Optical flow cytometers that combine fluorescence and scatter measurements present significant mechanical challenges due to the very limited space available to mount all of the optics along the three different walls of the flow cell.

[0024] As a prior art, Patent Document 1 discloses an epifluorescence optical flow cytometer that uses the same lens for focusing the excitation beam and detecting the fluorescence.

[0025] However, regardless of the fluorescence measurement direction (90 degrees, epifluorescence, etc.), a high NA lens is still required to collect the fluorescence. This creates strong mechanical constraints around the flow cell, and there is not enough space for multiple high NA optics. [Prior art documents] [Patent documents]

[0026] [Patent Document 1] International Publication No. 2019 / 058152 Summary of the Invention [Problem to be solved by the invention]

[0027] The object of the present invention is to propose a compact optical flow cytometer for performing fluorescence and scatter measurements.

[0028] Another object of the present invention is to eliminate the need for high NA forward scatter optics in optical flow cytometers that combine fluorescence and forward scatter measurements.

[0029] The present invention also aims to ensure consistency in forward scatter measurements.

[0030] The present invention also aims to resolve mechanical conflicts around the flow cell. [Means for solving the problem]

[0031] At least one of the above objects is an optical flow cytometer for fluorescence and scatter measurements, comprising: a non-coherent light source intended to generate a radiation beam; a beam truncation device, a first pass for passing a first portion of the illumination beam at a first divergence angle, the first portion being for scatter measurements; a second passage for passing a second portion of the illumination beam at a second divergence angle, the second portion being for fluorescence measurement, the second divergence angle being greater than the first divergence angle, the first passage and the second passage being separated by a region of the beam truncation device that blocks the illumination beam; a beam truncation device having at least - at least one focusing lens for focusing the first and second portions of the illumination beam onto a flow cell including an optical interrogation zone; a flow cell intended to contain particles flowing through an optical interrogation area; a scatter detector that receives light scattered from the first portion of the illumination beam as the particle traverses the optical interrogation area; - a fluorescence detector that receives fluorescence emitted by particles passing through the optical inspection area; This is achieved with an optical flow cytometer comprising:

[0032] The beam truncation device according to the present invention is not a beam splitter. A beam truncation device is used to modify the cross-section of an illumination beam by blocking part of the cross-section. The cross-sectional shapes of the input and output beams are not the same. Conversely, with a beam splitter, the input and output beams have the same cross-sectional shapes.

[0033] A first portion of the illumination beam is intended to illuminate the suspended particles so that forward scattered light about the optical axis can be accurately measured.

[0034] The second portion of the illumination beam is for illuminating the suspended particles from one or more directions different from the direction of the first portion of the illumination beam. In this configuration according to the invention, a gap is provided between the first portion of the illumination beam and the second portion of the illumination beam. This gap is advantageously used to collect scattered light.

[0035] Using an optical flow cytometer according to the present invention, scattering measurements are more accurate than those of the prior art because detection is performed below 5° (measured from a semi-cone of the divergence angle of the illumination beam) without contaminating the second beam provided for fluorescence excitation. Accurate detection of the forward scattering range defined by [0°, +5°] (measured from a semi-cone of the divergence angle of the illumination beam) allows for accurate estimation of the size of particles within a sample.

[0036] The first and second portions of the illumination beam are separated at an angle, allowing forward scattered light to be measured without disturbing the second portion of the beam, which is intended for fluorescence excitation. Therefore, high numerical aperture (NA) optics are not required for forward scattered light measurements. The present invention relaxes the mechanical constraints around the flow cell and reduces the cost of the optics for collecting forward scattered light. In fact, the optics used for scatter measurements can be smaller than those used in the prior art.

[0037] According to the present invention, scattering measurements may be performed simultaneously with fluorescence measurements.

[0038] According to the present invention, the non-coherent light source may be an LED, a filament lamp or an arc lamp.

[0039] Separate light sources of different wavelengths may be used, for example, incoherent extended light sources may be used at ultraviolet, visible, or infrared wavelengths.

[0040] According to the invention, the cross section of the second passage may be larger than the cross section of the first passage, so that the energy of the second portion of the illumination beam is greater than the energy of the first portion of the illumination beam.

[0041] The first pass passes a low divergence portion of the illumination beam, and the second pass passes a high divergence portion of the illumination beam.

[0042] The divergence angle is considered to be the maximum angle of inclination of the rays that make up the beam. The low divergence portion of the illumination beam is closer to the optical axis than the high divergence portion of the illumination beam.

[0043] The present invention allows for the separation of the high energy, high divergence illumination beam required for excitation of fluorescent dyes from the low energy, low divergence illumination beam required for forward scatter measurements.

[0044] A low divergence beam is particularly advantageous for forward scatter measurements because it does not require high NA optics, so the optics that collect the forward scattered light can be located away from the flow cell and lower NA optics can be used, reducing the cost of the optics.

[0045] According to a preferred embodiment of the present invention, the first passage may be, for example, circular and centrally located along the optical axis of the illumination beam.

[0046] The second passageway may be circular and concentric with the first passageway.

[0047] According to one embodiment of the present invention, a second beam truncation device may be positioned after the flow cell relative to the light propagation axis to block the first and / or second portions of the illumination beam.

[0048] The second beam truncation device may have an aperture whose sole purpose is to pass all or part of the scattered light.

[0049] According to a preferred embodiment of the present invention, when the first passage has a circular cross section with a radius R, the radial distance between the first passage and the second passage may be equal to or greater than 0.2×R.

[0050] For example, if the first and second passages are circular and concentric, the dimensions may be as follows: Radius of first passage: [0;R] Radius of the block: [R; n × R], n>1.2 Radius of the second passage: [n × R; Rmax]

[0051] Preferably, the cross section of the first passage is a fraction of the cross section of the second passage, thereby producing a high power, high divergence beam for fluorescence measurements and another low energy, low divergence beam for coherent forward scatter measurements.

[0052] According to the present invention, the beam truncation device may be located on either side of the focalization lens. If the focalization lens is a lens group, the beam truncation device may be located within the lens group. The beam truncation device does not have to be located close to the focalization lens. Multiple beam truncation devices may be used in different locations.

[0053] According to the present invention, the beam truncation device may comprise at least one filter disposed in the first path and / or the second path to change the spectral characteristics of the first portion of the illumination beam and / or the second portion of the illumination beam, respectively.

[0054] The type of filter can be colored glass (such as Schott BG12) or an interference filter (such as Semrock 447 / 60nm BrightLine).

[0055] According to the invention, the beam truncation device may comprise at least one polarizer disposed in the first path and / or the second path for changing the polarization of the first portion of the illumination beam and / or the second portion of the illumination beam, respectively.

[0056] Polarizers can be linear (such as EdmundOptics Linear Polarizing Film model number 19-003), or circular (such as Edmund Optics Circular Polarizer model number 88-095), or corrugated (such as EdmundOptics model number 91-012).

[0057] According to the invention, the beam truncation device may comprise at least one further lens located in the first path and / or the second path for changing the direction of the first portion of the illumination beam and / or the second portion of the illumination beam, respectively.

[0058] For example, a small plano-convex lens, such as an EdmundOptics model number 49-173, may be placed in the first passage.

[0059] The filters, polarizers and directional lenses of the beam truncation device are removable.

[0060] According to a preferred embodiment of the present invention, the beam truncation device may comprise or consist of a diaphragm in which the first and second passages are holes.

[0061] The diaphragm may be located near the focusing lens of the illumination beam, realizing the splitting by two holes, which make it possible to distinguish between two beams: - a first low-divergence, low-energy beam used for forward scattering measurements, and - A second, highly divergent, high-energy beam used to excite the fluorescent dye.

[0062] According to one embodiment of the present invention, the first and / or second beam truncation device may be a light blocking material deposited on the surface of the lens.

[0063] According to one embodiment of the present invention, the cross section of the first passageway and / or the second passageway may be circular, square, rectangular or any shape.

[0064] According to an embodiment of the present invention, the cross section of the first passage and / or the second passage may or may not be centered relative to the optical axis.

[0065] According to the present invention, the focusing lens may be a single lens or a group of lenses.

[0066] According to one embodiment of the present invention, the focusing lens may be designed to also collect the fluorescence coming from the flow cell, which is detected by the fluorescence detector to provide a measurement of epi-fluorescence.

[0067] In this embodiment, a focusing lens is used to focus the illumination beam into the sample and collect the fluorescence coming from the sample.

[0068] According to another embodiment of the invention, a fluorescence lens is positioned at 90° to the optical axis to focus the fluorescence coming from the flow cell onto the fluorescence detector for 90° fluorescence measurements.

[0069] The present invention may advantageously include fluorescence detection at 90 degrees or epi-fluorescence, in either case the present invention ensures consistent forward scatter measurements.

[0070] In another embodiment of the invention, a light collection module may be placed after the flow cell to detect forward scattered light, mid-angle scattered light or axial light loss.

[0071] According to a preferred embodiment of the present invention, the scatter detector may be arranged to detect light scattered by the airborne particles at angles in the range [0°, 5°] measured from the divergence angle of the first portion of the illumination beam. Other angle ranges may be used, such as [0°, 10°] or [0°, 15°].

[0072] If the divergence angle of the illumination beam is too large, the light scattered at low angles relative to the illumination beam is highly correlated with particle size, so the accuracy of blood cell characterization by optical transducers is significantly reduced when the illumination beam is highly divergent.

[0073] The present invention is based on a beam segmentation stop that produces a high power, high divergence beam for fluorescence measurements and a low energy, low divergence beam for forward scatter measurements.

[0074] According to one embodiment of the present invention, a detection lens or group of lenses may be placed after the flow cell to collect and focus the light scattered by the suspended particles onto a scatter detector.

[0075] The scatter detector may be placed directly at or near the optical interrogation area to directly detect scattered light.

[0076] For the purpose of illustrating the invention, there is shown in the drawings a form that is presently preferred; it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities. [Brief explanation of the drawings]

[0077] [Figure 1] 1 is an overall view showing the components of an optical flow cytometer according to the present invention; [Figure 2] 1 is a schematic diagram showing the arrangement of optical components and light trajectories in an example of an optical flow cytometer according to the present invention. [Figure 3] 1 is a schematic front view of a beam truncation device according to the present invention; [Figure 4] FIG. 2 is a schematic front view of another beam truncation device according to the present invention. [Figure 5] 1 is a schematic diagram illustrating the action of a beam truncation device on an illumination beam. [Figure 6] FIG. 1 is a schematic diagram of low angle scatter detection. [Figure 7] 7 is a cross-sectional view of a first portion of the scattered light and illumination beam of FIG. 6; DETAILED DESCRIPTION OF THE INVENTION

[0078] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawings and the accompanying detailed description are not intended to limit the invention to the particular forms disclosed, but rather the invention is intended to cover all modifications, equivalents, and alternatives falling within the scope of the invention as defined by the appended claims.

[0079] Exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings, in which like reference numerals refer to like elements, and in which:

[0080] FIG. 1 is an overall diagram showing components of an optical flow cytometer in accordance with an exemplary embodiment of the present invention.

[0081] Referring to Figure 1, an optical flow cytometer 1 of the present invention includes an illumination module 2 that generates two illumination beams 3a and 3b (from a single, incoherent light source) toward a flow cell 4 in which particles, such as blood cells, are suspended. The optical flow cytometer 1 includes motorized and / or other means for causing the sample cells or blood cells to flow and focus in a stream (which may or may not be surrounded by a sheath fluid). The blood cells circulate within the flow cell 4.

[0082] The illumination beams 3a and 3b are focused and directed perpendicularly across the cell stream, inducing fluorescence of the fluorescent sample particles or their markers. The fluorescence 7 produced by the blood cells is collected by the fluorescence measurement module 5.

[0083] The illumination beams 3a and 3b also induce scattered light 8 as the blood cells pass through the optical interrogation zone. The optical flow cytometer 1 comprises a scatter measurement module 6 provided to collect scattered light 8 coming from the flow cell 4.

[0084] A processor unit 9 is provided for controlling the excitation module 2 for the excitation signal, which also controls the fluorescence measurement module 5 and the scatter measurement module 6 for detecting the direct and / or indirect scatter signals.

[0085] In the embodiment of Figure 1, the scatter measurement module 6 is positioned opposite the illumination module 2, and the fluorescence measurement module 5 is positioned at 90° from the optical axis of the illumination beam. Other arrangements are possible by providing mirrors, lenses and / or beam splitters to deflect the light.

[0086] 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 such a configuration, the same focusing lens is used to focus the excitation beam onto the flow cell and to collect the fluorescence from the flow cell.

[0087] According to the invention, the illumination beam is split into a first excitation beam portion 3a and a second excitation beam portion 3b.

[0088] The first portion 3a is preferably a low energy and low divergence beam.

[0089] The second portion 3b is preferably a high energy and highly divergent beam.

[0090] The two beams 3a and 3b reach the sample from different directions, so that the first part 3a and the second part 3b travel in different directions after passing through the sample.

[0091] In the present invention, after passing through the sample, there is a gap between the first portion 3a of the illumination beam and the second portion 3b of the illumination beam. Therefore, this gap is used to detect scattered light. This detection is considered to be consistent because there is no interfering light of the second portion 3b of the excitation beam.

[0092] For example, scattering measurements within the range [0°, 5°] (from the half-cone angle of the divergence angle of the illumination beam) can be made within this gap. In fact, this 5° is considered to exceed the cross-sectional area of ​​the first portion 3a of the illumination beam. Figure 6 shows that 5° is detected in the gap without the excitation beam of the second portion 3b. In this way, the scattering measurement has improved accuracy. Figure 7 is a cross-sectional view of the scattered light and first portion of the illumination beam of Figure 6. The first portion 3a of the illumination beam is a circle surrounded by a coronal portion representing light scattered at 5°.

[0093] FIG. 2 shows details of the optical system of an optical flow cytometer.

[0094] The module 2 comprises an incoherent extended light source, such as an LED, that emits a 485 nm illumination beam 3. The beam truncation device 11 has at least two paths for blocking a portion of the excitation beam 3 and for passing two sub-beams (a first portion of the illumination beam 3a and a second portion of the illumination beam 3b).

[0095] 3 shows an example of a beam truncation device according to the present invention. The device has two passages, holes, or transparent (at the excitation wavelength) materials 11a and 11b that allow a first portion of the illumination beam 3a and a second portion of the illumination beam 3b to pass through, respectively. The remaining light of the excitation beam is blocked by an opaque material 11c.

[0096] The first passage 11A is a circular hole in the center of the beam truncation device, so that the illumination axial light passes through this first passage 11a and constitutes the first portion 3a of the illumination beam.

[0097] The second passage 11b has an annular shape concentric with the first passage. The cross-sectional area of ​​the second passage is larger than that of the first passage. The second portion 3b of the illumination beam contains a thicker light beam than the first portion 3a, which means it has higher energy. Because the center of the second passage is within the first passage, the second portion 3b of the illumination beam diverges more than the first portion 3a of the excitation beam.

[0098] Figure 4 shows another illumination of the beam truncation device, with different cross-sectional areas of the passages. The first passage 11A is an oval hole extending outward from the center of the beam truncation device. The second passage 11b is a square hole separated from the first passage.

[0099] The beam truncation device according to the invention is not a beam splitter: a standard beam splitter does not block any of the input beam and can be used with any kind of light source (coherent or not).

[0100] Conversely, the beam truncation device of the present invention utilizes the interruption of an input beam to change its cross-sectional shape. It is well known to those skilled in the art of optics that when a coherent light source is used, the interruption of the beam results in a strong diffraction pattern. Therefore, the beam truncation device of the present invention is a split aperture and is not suitable for use with coherent light sources such as lasers.

[0101] Returning to Figure 2, the first portion of the illumination beam 3a and the second portion of the illumination beam 3b are focused using a lens 12 onto an optical interrogation area 13 inside the flow cell 4. The sample is intended to circulate within the optical interrogation area.

[0102] After the flow cell, both the first portion 3a and the second portion 3b of the illumination beam are blocked by a second truncation device 14. The device 14 has an annular hole 21 that allows light 20 scattered by the particles to reach a detector 16.

[0103] The second portion 3b of the illumination beam is strongly divergent, so its angular position is far enough away from the annular hole of the second truncation device 14. Because the scattered intensity falls off rapidly with angle, the portion 3b of the illumination beam passes through the annular hole of the second truncation device 14 and produces only a very low scattered intensity.

[0104] Conversely, the portion 3a of the illumination beam is very close to the annular hole of the second truncation device 14. Therefore, the scattering contribution of the portion 3a of the illumination beam passing through the annular hole of the second truncation device 14 is very large.

[0105] As a result, although portion 3b of the illumination beam has a much stronger power than 3a, this portion 3b is farther away from the annular hole of the second truncation device 14 and therefore does not make a significant scattering contribution when passing through the annular hole.

[0106] A detection lens 15 is used to direct the light scattered by the particles onto a photodetector 16 .

[0107] Lens 15 and photodetector 16 are components of scatterometry module 6 of FIG.

[0108] Fluorescence is generated by passage of the first portion 3a of the excitation beam and the second portion 3b of the excitation beam through the optical interrogation area 13. A fluorescence lens 17 focuses a portion 18 of the fluorescent light onto a detector 19.

[0109] Fluorescence lens 17 and detector 19 are components of fluorescence measurement module 5 of FIG.

[0110] In the configuration of the present invention, the detection lens 15 can be small, which means there are fewer mechanical constraints on mounting the large (high numerical aperture) focusing lens 12 and fluorescence lens 17.

[0111] Figure 5 shows the splitting of the illumination beam in detail. A first portion 3a of the excitation beam and a second portion 3b of the illumination beam are produced from a single light source. A focusing lens causes the first portion 3a and the second portion 3b to all converge within the flow cell, thereby creating the optical interrogation zone.

[0112] After focusing into the sample, the first portion 3a remains separated from the second portion 3b.

[0113] The proposed invention eliminates the need for high NA optics for forward scatter measurements, thereby easing mechanical constraints around the flow cell and reducing the cost of the forward scatter collection optics.

[0114] In the most efficient setup, the beam dedicated to scattering is the central beam with low divergence, but the principles of the proposed invention also apply when the beam dedicated to scattering is not the central beam.

[0115] The present invention allows for improved scatter measurements by detecting scattered light that is not obstructed by the high intensity fluorescence excitation beam 3b. This improves the quality of the scatter measurement and allows for the use of small lenses to receive the scattered light. This allows for the placement of large high numerical aperture lenses around the flow cell for focusing the illumination beam and other large high numerical aperture lenses for fluorescence detection.

[0116] By reducing the size of the lens that collects the scattered light, space limitations for other lenses that focus the illumination and collect the fluorescent light are eliminated.

[0117] Numerous variations and modifications will become apparent to those skilled in the art once the above disclosure is fully appreciated, and it is intended that the following claims be interpreted to embrace all such variations and modifications.

Claims

1. 1. An optical flow cytometer for fluorescence and scatter measurements, comprising: a non-coherent light source intended to generate a radiation beam; a beam truncation device, a first pass for passing a first portion of said illumination beam at a first divergence angle, said first portion being for scatterometry measurements; a second path for passing a second portion of the illumination beam at a second divergence angle, the second portion being for fluorescence measurement, the second divergence angle being greater than the first divergence angle, the first path and the second path being separated by a region of the beam truncation device that blocks the illumination beam; a beam truncation device having at least at least one focusing lens for focusing said first and second portions of said illumination beam onto a flow cell containing an optical interrogation zone; said flow cell intended to contain particles flowing through said optical interrogation zone; a scatter detector for receiving light scattered from said first portion of said illumination beam as a particle traverses said optical interrogation area; a fluorescence detector for receiving fluorescence emitted by particles traversing said optical interrogation area; An optical flow cytometer comprising:

2. 2. The optical flow cytometer of claim 1, wherein the non-coherent light source is an LED, a filament lamp, or an arc lamp.

3. 3. The optical flow cytometer according to claim 1, wherein the cross section of the second passage is larger than the cross section of the first passage.

4. 4. An optical flow cytometer according to claim 1, wherein the first passage is centrally located along the optical axis of the illumination beam.

5. 5. The optical flow cytometer of claim 1, wherein a second beam truncation device is arranged after the flow cell to block the first and / or second portions of the illumination beam.

6. 6. An optical flow cytometer according to claim 1, wherein when the first passage has a circular cross section with a radius R, the radial distance between the first passage and the second passage is 0.2×R or greater.

7. 7. The optical flow cytometer of claim 1, wherein the beam truncation device comprises at least one filter disposed in the first path and / or the second path to change the spectral characteristics of the first portion of the illumination beam and / or the second portion of the illumination beam, respectively.

8. 8. The optical flow cytometer of claim 1, wherein the beam truncation device comprises at least one polarizer arranged in the first path and / or the second path to change the polarization of the first portion of the illumination beam and / or the second portion of the illumination beam, respectively.

9. 9. The optical flow cytometer of claim 1, wherein the beam truncation device comprises at least one further lens located in the first path and / or the second path to change the direction of the first portion of the illumination beam and / or the second portion of the illumination beam, respectively.

10. 10. The optical flow cytometer according to claim 1, wherein the beam truncation device has a diaphragm 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, wherein the beam truncation device is a light blocking material deposited on the surface of a lens.

12. 12. The optical flow cytometer according to claim 1, wherein the cross section of the first passage and / or the second passage is circular, square, rectangular or of any shape.

13. 13. The optical flow cytometer according to claim 1, wherein the focusing lens is designed to collect fluorescence coming from the flow cell, and the fluorescence coming from the flow cell is detected by the fluorescence detector to realize a measurement of epifluorescence.

14. 13. An optical flow cytometer according to any one of claims 1 to 12, characterized in that a fluorescence lens is arranged at 90° to the optical axis to focus the fluorescence coming from the flow cell onto the fluorescence detector for performing a 90° fluorescence measurement.

15. 14. An optical flow cytometer according to any one of claims 1 to 13, characterized in that the scatter detector is positioned to detect light scattered by suspended particles at angles in the range [0°, 5°] measured from the divergence angle of the first portion of the illumination beam.

16. 16. An optical flow cytometer according to any one of claims 1 to 15, characterized in that a detection lens or group of lenses is positioned after the flow cell to collect and focus light scattered by the suspended particles onto the scatter detector.

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