An optical flow cytometer measures fluorescence and scattering by splitting a beam emitted from a single non-coherent 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 performing fluorescence measurement and scattering measurement on the flow of blood cells. The present invention relates to the field of automatic apparatuses for counting and characterizing particles suspended in a liquid medium, and more specifically, to the field of hematological instruments for counting and characterizing various types of cells such as leukocytes, erythrocytes, and platelets contained in blood samples, as well as other types of particles such as algae and bacteria.

[0002] Accordingly, the prior art and the proposed present invention relate to an apparatus for counting and characterizing various types of cells contained in a blood sample, and measuring the relative distribution thereof within various subpopulations based on light scattering and fluorescence.

Background Art

[0003] In optical flow cytometers, it is known to use an LED light source to perform scattering measurement and fluorescence measurement.

[0004] Scattering measurement in such a configuration involves several limitations.

[0005] Unlike fluorescence, scattering from a single cell is extremely highly anisotropic. Scattering measurement of a single cell depends not only on the parameters of the cell to be measured, but also on the shape of the illumination beam.

[0006] It is also known that the intensity of light scattered by a single cell attenuates rapidly as the scattering angle increases, and reflects cell parameters that vary greatly depending on the angle of the focused scattered light beam.

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

[0008] Furthermore, it is obvious to those skilled in the art that scattered light can only be measured if it exceeds the divergence angle of the irradiated beam. If the divergence angle of the semicone of the irradiated beam is α (alpha) with respect to the optical axis, the scattered light must be collected from an angle greater than or equal to α (alpha). Otherwise, a portion of the irradiated beam will also be collected. Therefore, the scattering angle is determined not by the optical axis, but by the semicone of the divergence angle of the irradiated beam.

[0009] Single-cell scattering exhibits strong anisotropy; for example, the light intensity of small-angle scattering (measured from a semicone of the divergence angle of the irradiation beam) is very strong at angles less than 10 degrees. Regardless of the light source, the photodetector does not require special sensitivity. Standard photodiodes are common in flow cytometry optics for measuring small-angle scattering. The intensity of the irradiation beam is not a critical parameter for measuring small-angle scattering from single blood cells.

[0010] Therefore, the direction in which scattered light from a single cell is collected is, - Divergence angle of the irradiation beam, and - Cell parameters to be measured (size or complexity) It depends on [something].

[0011] Fluorescence measurements also have several limitations.

[0012] Flow cytometry sometimes relies heavily on fluorescence measurements to characterize blood cells in detail. Dyes bind to specific cellular characteristics (such as nucleic acid content) and emit fluorescence when irradiated at the appropriate wavelength.

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

[0014] The fluorescence emission intensity is proportional to the excitation (or irradiation) intensity of the dye. Therefore, it is advantageous to increase the excitation (or irradiation) intensity as much as possible to maximize fluorescence emission.

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

[0016] Fluorescence is known to be isotropic emission. Therefore, the results will be nearly the same regardless of the direction of fluorescence collection.

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

[0018] The use of non-coherent extended light sources also comes with several limitations.

[0019] Unlike lasers, high-power LEDs are highly divergent, extended light sources. Therefore, a high numerical aperture (NA) optical system is required to maximize the light collection efficiency. Flow cytometry optical systems must focus the excitation beam onto the flow cell (inspection area), so a high numerical aperture (NA) focusing optical system is also required to handle highly divergent beams.

[0020] Therefore, the limitations of LEDs are based on the following highly divergent irradiation (i.e., excitation) beam. • High NA focusing lens, and • High NA scattering collection lens.

[0021] To maximize the irradiation (excitation) of fluorescent dyes, it is essential to use high-NA optics for both collecting and focusing the light from the LED light source. Since the greatly diverged excitation beam passes through the flow cell, high-NA optics are also necessary to measure the forward scattered light. This is because the scattering must be measured beyond the divergence angle of the irradiation beam.

[0022] Since a high-NA optical system is also required for fluorescence measurement, a high-NA optical system is required for all optical functions including light source focusing, forward scattered light (FSC) measurement, and fluorescence measurement.

[0023] In an optical flow cytometer combining fluorescence measurement and scattering measurement, the space for fixing all optical systems along three different walls of the flow cell is very limited, which makes mechanical design extremely difficult.

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

[0025] However, regardless of the fluorescence measurement direction (90 degrees, epifluorescence, etc.), a high-NA lens is still required to collect fluorescence. For this reason, strong mechanical constraints arise around the flow cell, and there is insufficient space for a plurality of high-NA optical systems.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0026]

Patent Document 1

Summary of the Invention

Problem to be Solved by the Invention

[0027] An object of the present invention is to provide a compact optical flow cytometer for performing fluorescence measurement and scattering measurement.

[0028] Another object of the present invention is to eliminate the need for a high N.A. forward scattering optical system in an optical flow cytometer combining fluorescence measurement and forward scattering measurement.

[0029] The present invention also aims to ensure the consistency of forward scattering measurement.

[0030] This invention also aims to resolve mechanical inconsistencies around the flow cell. [Means for solving the problem]

[0031] At least one of the above objectives is an optical flow cytometer for fluorescence measurement and scattering measurement, - A non-coherent light source intended to generate an illumination beam, - A beam truncation device, - A first passage for passing a first portion of the irradiation beam at a first divergence angle, wherein the first portion is for scattering measurement, the first passage, - A second passage for passing a second portion of the irradiation beam at a second divergence angle, the second portion being for fluorescence measurement, the second divergence angle being greater than the first divergence angle, and the first and second passages being separated by the region of a beam truncation device that blocks the irradiation beam. A beam truncation apparatus having at least the following, - At least one focusing lens that focuses the first and second portions of the irradiation beam onto a flow cell including an optical inspection area, - A flow cell intended to contain particles flowing through the light inspection area, - A scattering detector that receives light scattered from a first portion of the irradiation beam as a particle crosses the optical inspection area, - A fluorescence detector that accepts fluorescence emitted by particles crossing the light inspection area and This is achieved with an optical flow cytometer equipped with [specific features / features].

[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 irradiated beam by blocking a portion of its cross-section. The cross-sectional shapes of the input beam and the output beam are not the same. Conversely, when using a beam splitter, the input beam and the output beam have the same cross-sectional shape.

[0033] The first portion of the irradiation beam is intended to irradiate suspended particles, particularly to allow for accurate measurement of forward scattered light around the optical axis.

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

[0035] Using the optical flow cytometer according to the present invention, detection of angles less than 5° (measured from the semicone of the irradiation beam's divergence angle) is performed without contaminating the second beam supplied for fluorescence excitation, resulting in more accurate scattering measurements than prior art. By accurately detecting the forward scattering range defined by [0°, +5°] (measured from the semicone of the irradiation beam's divergence angle), it becomes possible to accurately estimate the size of particles in the sample.

[0036] Because the first and second portions of the illumination beam are separated at an angle, it becomes possible to measure the forward scattered light without disturbing the second portion of the beam, which is intended for fluorescence excitation. Therefore, a high numerical aperture (NA) optical system is not required for forward scattering measurements. This invention alleviates the mechanical constraints around the flow cell and reduces the cost of the optical system for collecting forward scattered light. In fact, the optical system used for scattering 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 with different wavelengths may be used. For example, non-coherent extended light sources may be used at ultraviolet, visible, or infrared wavelengths.

[0040] According to the present invention, the cross-section of the second passage may be larger than the cross-section of the first passage. Therefore, the energy of the second portion of the irradiation beam is greater than the energy of the first portion of the irradiation beam.

[0041] The first passage allows the low-divergence portion of the irradiation beam to pass through, and the second passage allows the high-divergence portion of the irradiation beam to pass through.

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

[0043] The present invention makes it possible to separate the high-energy, high-divergence irradiation beam required for exciting fluorescent dyes from the low-energy, low-divergence irradiation beam required for forward scattering measurements.

[0044] Low-divergence beams are particularly advantageous for forward scattering measurements because they do not require high-NA optics. This allows the optics that collect the forward-scattered light to be moved away from the flow cell, enabling the use of low-NA optics and thus 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 irradiation beam.

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

[0047] According to one embodiment of the present invention, a second beam truncation device may be positioned behind the flow cell with respect to the optical propagation axis in order to block a first portion and / or a second portion of the irradiation beam.

[0048] The second beam truncation device may have holes intended solely for the purpose of allowing all or part of the scattered light to pass through.

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

[0050] For example, if the first and second passages are circular and concentric, the dimensions may be as follows: Radius of the first passage: [0;R] Radius of the block area: [R; n × R] where 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 generating a high-power, high-divergence beam for fluorescence measurements and another low-energy, low-divergence beam for consistent forward scattering measurements.

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

[0053] According to the present invention, the beam truncation device may include at least one filter installed in the first and / or second passages to change the spectral characteristics of the first and / or second portions of the irradiation beam, respectively.

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

[0055] According to the present invention, the beam truncation device may include at least one polarizer installed in the first and / or second passages to change the polarization of the first portion and / or the second portion of the irradiation beam, respectively.

[0056] Polarizers can be linear (e.g., Edmund Optics Linear Polarizing Film, model number: 19-003), circular (e.g., Edmund Optics Cicular Polarizer, model number: 88-095), or corrugated (e.g., Edmund Optics, model number: 91-012).

[0057] According to the present invention, the beam truncation device may include at least one additional lens installed in the first and / or second passages to change the direction of the first portion and / or the second portion of the irradiation beam, respectively.

[0058] For example, a small plano-convex lens, such as Edmund Optics model number 49-173, may be installed 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 have or be composed of a first passage and a second passage which are holes.

[0061] The aperture achieves division through two holes and may be located near the focusing lens of the illumination beam. These two holes make it possible to distinguish between the following two beams: - A first low-divergence, low-energy beam used for forward scattering measurements, and - A second high-divergence, high-energy beam used to excite fluorescent dyes.

[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 a lens.

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

[0064] According to one embodiment of the present invention, the cross-sections of the first passage and / or the second passage may or may not be centered with respect 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 fluorescence coming from the flow cell. This fluorescence coming from the flow cell is detected by a fluorescence detector to perform epifluorescence measurement.

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

[0068] According to another embodiment of the present invention, the fluorescent lens is positioned at a 90° angle to the optical axis to focus the fluorescence coming from the flow cell onto the fluorescence detector in order to perform a 90° fluorescence measurement.

[0069] The present invention may, advantageously, include fluorescence detection or epifluorescence at 90 degrees. In either case, the present invention ensures consistent forward scattering measurements.

[0070] In another embodiment of the present invention, a light collection module may be placed behind 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 scattering detector may be positioned to detect light scattered by suspended particles at angles within the range of [0°, 5°], measured from the divergence angle of a first portion of the irradiation beam. Other angular ranges such as [0°, 10°] or [0°, 15°] may be used.

[0072] When the divergence angle of the irradiation beam is too large, the light scattered at low angles relative to the irradiation beam is closely related to the particle size. Therefore, when the irradiation beam diverges significantly, the accuracy of blood cell characteristic evaluation using an optical transducer decreases considerably.

[0073] This invention is based on a beam segmentation stop that generates a high-power, high-divergence beam for fluorescence measurements and a low-energy, low-divergence beam for forward scattering measurements.

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

[0075] The scattering detector may be placed directly in or near the optical inspection area to directly detect scattered light.

[0076] While preferred embodiments are shown in the drawings to illustrate the present invention, it is understood that the present invention is not limited to these exact configurations and means. [Brief explanation of the drawing]

[0077] [Figure 1] This is an overall diagram showing the components of the optical flow cytometer according to the present invention. [Figure 2] This is a schematic diagram showing the arrangement of optical components and the trajectory of light in an example of an optical flow cytometer according to the present invention. [Figure 3] This is a schematic front view of the beam truncation apparatus according to the present invention. [Figure 4] This is a schematic front view of another beam truncation apparatus according to the present invention. [Figure 5] This is a schematic diagram showing the effect of the beam truncation device on the irradiation beam. [Figure 6] This is a schematic diagram of low-angle scattering detection. [Figure 7] Figure 6 shows the scattered light and a cross-sectional view of the first portion of the irradiation beam. [Modes for carrying out the invention]

[0078] While various modifications and alternative forms are possible for the present invention, specific embodiments are shown as examples in the drawings and described in detail herein. However, it should be understood that the drawings and their detailed descriptions are not intended to limit the present invention to any particular form disclosed, but rather that the present invention is intended to encompass all modifications, equivalents, and alternative forms that fall within the scope of the present invention as described in the appended claims.

[0079] Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. Similar reference numerals in the drawings refer to similar elements.

[0080] Figure 1 is an overall view showing the components of an optical flow cytometer according to an exemplary embodiment of the present invention.

[0081] 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 (from a single non-coherent light source) toward a flow cell 4 in which particles such as blood cells are suspended. The optical flow cytometer 1 has motorized and / or other means for moving and focusing sample cells or blood cells into the flow (which may or may not be surrounded by sheath fluid). The blood cells circulate within the flow cell 4.

[0082] The irradiation beams 3a and 3b are focused and directed perpendicularly across the flow of cells to induce fluorescence of fluorescent sample particles or their markers. The fluorescence 7 produced by the blood cells is collected by the fluorescence measurement module 5.

[0083] The irradiation beams 3a and 3b also induce scattered light 8 as blood cells pass through the photo-examination area. The optical flow cytometer 1 has a scattering measurement module 6 provided for collecting scattered light 8 coming from the flow cell 4.

[0084] A processor unit 9 is provided to control the excitation module 2 for the excitation signal. The processor unit 9 also controls the fluorescence measurement module 5 and the scattering measurement module 6 to detect direct and / or indirect scattering signals.

[0085] In the embodiment shown in Figure 1, the scattering measurement module 6 is positioned on the opposite side of the irradiation module 2, and the fluorescence measurement module 5 is positioned at 90° from the optical axis of the irradiation beam. Other arrangements are possible by deflecting the light with mirrors, lenses and / or beam splitters.

[0086] The present invention also relates to epifluorescence configurations 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 fluorescence is collected from the flow cell.

[0087] According to the present invention, the irradiation beam is divided into a first portion 3a of the excitation beam and a second portion 3b of the excitation beam.

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

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

[0090] Since the two beams 3a and 3b reach the sample from different directions, the first portion 3a and the second portion 3b proceed in different directions after passing through the sample.

[0091] In this invention, after passing through the sample, there is a gap between the first portion 3a and the second portion 3b of the irradiation beam. Therefore, scattered light is detected using this gap. Since there is no interfering light from the second portion 3b of the excitation beam, this detection is considered to be consistent.

[0092] For example, scattering measurements within the range of [0°, 5°] (from the semiconical angle of the divergence angle of the irradiation beam) can be performed within this gap. In fact, this 5° is considered to exceed the cross-sectional area of ​​the first portion 3a of the irradiation beam. Figure 6 shows that 5° was detected in the gap where there is no excitation beam in the second portion 3b. Thus, the accuracy of the scattering measurement has improved. Figure 7 is a cross-sectional view of the scattered light of the irradiation beam and the first portion in Figure 6. The first portion 3a of the irradiation beam is a circle surrounded by a coronal portion representing the light scattered at 5°.

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

[0094] Module 2 has a non-coherent extension light source, such as an LED, that emits a 485 nm irradiation beam 3. The beam truncation device 11 has at least two passages for blocking a portion of the excitation beam 3 and allowing two sub-beams (a first portion 3a of the irradiation beam and a second portion 3b of the irradiation beam) to pass through.

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

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

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

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

[0099] The beam truncation device according to the present invention is not a beam splitter. A standard beam splitter does not interrupt the input beam in any way and can be used with any type of light source (whether coherent or not).

[0100] Conversely, the beam truncation device according to the present invention changes the cross-sectional shape of the input beam by utilizing the interruption of the beam. It is widely known to those skilled in the field of optics that when a coherent light source is used, beam interruption generates a strong diffraction pattern. Therefore, the beam truncation device according to the present invention is a segmented aperture and is not suitable for use with coherent light sources such as lasers.

[0101] Returning to Figure 2, the first portion 3a and the second portion 3b of the irradiation beam are focused into the photo-inspection area 13 inside the flow cell 4 using the lens 12. The sample is intended to circulate within the photo-inspection area.

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

[0103] Because the second portion 3b of the irradiation beam diverges strongly, its angular position is sufficiently far from the annular opening of the second truncation device 14. Since the scattering intensity decreases rapidly with angle, the portion 3b of the irradiation beam passes through the annular opening of the second truncation device 14 and produces only extremely low scattering intensity.

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

[0105] As a result, although portion 3b of the irradiated beam has a much stronger output than 3a, this portion 3b is far from the annular opening of the second truncation device 14, so its scattering contribution when passing through the annular opening is not large.

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

[0107] The lens 15 and the photodetector 16 are components of the scattering measurement module 6 shown in Figure 1.

[0108] Fluorescence is generated when the first portion 3a and the second portion 3b of the excitation beam pass through the photoinspection area 13. The fluorescence lens 17 focuses a portion of the fluorescence 18 onto the detector 19.

[0109] The fluorescent lens 17 and the detector 19 are components of the fluorescence measurement module 5 shown in Figure 1.

[0110] In the configuration of the present invention, the detection lens 15 may be small. This means that there are fewer mechanical constraints when installing large (high numerical aperture) focusing lens 12 and fluorescent lens 17.

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

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

[0113] The proposed invention eliminates the need for a high NA optical system for forward-scattered light measurements. This reduces mechanical constraints around the flow cell and lowers the cost of the forward-scattered light collection optical system.

[0114] In the most efficient setup, the beam dedicated to scattering is a low-divergence central beam. However, the principle of the proposed invention also applies when the beam dedicated to scattering is not the central beam.

[0115] This invention makes it possible to improve scattering measurements by detecting scattered light that is not interfered with by the high-intensity fluorescence excitation beam 3b. The quality of scattering measurements is improved, and scattered light can be received using a small lens. Therefore, it becomes possible to arrange a large lens with a high numerical aperture around the flow cell, using one lens for focusing the illumination beam and another large lens with a high numerical aperture for fluorescence detection.

[0116] By reducing the size of the lens that collects scattered light, the constraints on space for other lenses that focus the illumination and collect fluorescence are removed.

[0117] With a full understanding of the above disclosure, numerous variations and modifications will become apparent to those skilled in the art. The following claims shall be construed to encompass all such variations and modifications.

Claims

1. An optical flow cytometer for fluorescence measurement and scattering measurement, - A non-coherent light source (10) intended to generate an illumination beam (3), - A beam truncation device (11), - A first passage (11a) for passing a first portion (3a) of the illumination beam (3) at a first divergence angle, wherein the first portion (3a) is for scattering measurement, the first passage (11a), - A second passage (11b) for allowing a second portion (3b) of the illumination beam (3) to pass through at a second divergence angle, wherein the second portion (3b) is for fluorescence measurement, the second divergence angle is greater than the first divergence angle, and the first passage and the second passage are separated by a region of the beam truncation device that blocks the illumination beam. A beam truncation apparatus (11) having at least the following: - At least one focusing lens (12) that focuses the first portion (3a) and the second portion (3b) of the illumination beam onto a flow cell (4) including the light inspection area (13), - The flow cell (4) is intended to contain particles flowing through the light inspection area, and the beam truncation device (11) and the at least one focusing lens (12) are optically positioned between the light source and the flow cell (4), - A scattering detector (16) that receives light (20) scattered from the first portion (3a) of the illumination beam as a particle crosses the light inspection area, - A fluorescence detector (19) that receives fluorescence (18) emitted by particles crossing the aforementioned light inspection area and An optical flow cytometer equipped with the following features.

2. The non-coherent light source (10) is an LED, a filament lamp, or an arc lamp. The optical flow cytometer according to claim 1, characterized in that it is a lamp.

3. The optical flow cytometer according to claim 1, characterized in that the cross-section of the second passage (11b) is larger than the cross-section of the first passage (11a).

4. The optical flow cytometer according to claim 1, characterized in that the first passage (11a) is centrally located along the optical axis of the illumination beam.

5. The optical flow cytometer according to claim 1, characterized in that a second beam truncation device (14) is positioned behind the flow cell with respect to the propagation direction of the illumination beam (3) moving from the non-coherent light source (10) toward the flow cell in order to block the first and / or second portion of the illumination beam.

6. The optical flow cytometer according to claim 1, characterized in that when the first passage (11a) has a circular cross-section of radius R, the radial distance between the first passage and the second passage is 0.2 × R or more.

7. The optical flow cytometer according to claim 1, characterized in that the beam truncation device (11) includes at least one filter installed in the first passage (11a) and / or the second passage (11b) to change the spectral characteristics of the first portion and / or the second portion of the illumination beam, respectively.

8. The optical flow cytometer according to claim 1, characterized in that the beam truncation device (11) comprises at least one polarizer installed in the first passage (11a) and / or the second passage (11b) for changing the polarization of the first portion and / or the second portion of the illumination beam, respectively.

9. The optical flow cytometer according to claim 1, characterized in that the beam truncation device (11) comprises at least one additional lens installed in the first passage (11a) and / or the second passage (11b) for changing the direction of the first portion and / or the second portion of the illumination beam, respectively.

10. The optical flow cytometer according to claim 1, characterized in that the beam truncation device has an aperture through which the first passage and the second passage are holes.

11. The optical flow cytometer according to claim 1, characterized in that the beam truncation device (11) is a light-blocking material deposited on the surface of a lens.

12. The optical flow cytometer according to claim 1, characterized in that the cross-sections of the first passage (11a) and / or the second passage (11b) are circular, square, rectangular, or of any shape.

13. The optical flow cytometer according to claim 1, characterized in that the focusing lens (12) is designed to collect fluorescence coming from the flow cell (4), and the fluorescence coming from the flow cell is detected by the fluorescence detector to enable measurement of epifluorescence.

14. The optical flow cytometer according to claim 1, characterized in that the fluorescent lens (12) is positioned at a 90° angle to the optical axis of the illumination beam, and the fluorescence coming from the flow cell is focused onto the fluorescence detector in order to perform a 90° fluorescence measurement.

15. The optical flow cytometer according to claim 1, characterized in that the scattering detector (16) is arranged to detect light scattered by suspended particles at an angle within the range of [0°, 5°] measured from the divergence angle of the first portion of the illumination beam.

16. The optical flow cytometer according to claim 1, characterized in that, with respect to the propagation direction of the illumination beam (3) advancing from a non-coherent light source (10) toward the flow cell, a detection lens (15) or lens group is placed behind the flow cell to collect and focus the light scattered by suspended particles into the scattering detector.

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