JP7654195B1 - Particle Analyzers - Google Patents

Abstract

A particle imaging method capable of capturing high-quality images of particles flowing at high speeds in a particle analyzer equipped with a flow cytometer and a still image capturing function is provided.
[Solution] An imaging system is formed by an LED 21, a lens system 22 and a mirror 23, a flow cell 4, an objective lens 24 and a digital camera 25. This LED 21 is an incoherent LED, and high quality images are captured by emitting pulsed light with emission times in nanosecond units.
[Selected Figure] Figure 1

Description

The present invention relates to a particle analysis device equipped with a particle imaging function in a flow cytometer.

In a flow cytometer, a sample solution containing particles or cells is narrowed together with a sheath liquid to form a sheath flow in a flow cell. This sample solution is irradiated with a laser light, and the scattered light and fluorescent light emitted from the particles or cells are received and analyzed. This is a particle analysis device that is equipped with a function to capture transmitted light and fluorescent images of particles and cells in this flow cytometer.

JP 07-134093

JP 05-142137 A

Patent Publication 2017-116558

Patent Publication 2023-036596

Catalog "AImageStream▲R▼X Mark II", Merck Ltd., P2-P15

Eiichi Miyazaki, Kensei Okamoto: Prototype of a pulse light source using ultra-high brightness LED and its application, Transactions of the Society of Instrument and Control Engineers, Vol. 32, No. 3, 306/312 (1996)

The sample solution containing the particles is narrowed together with the sheath liquid to form a sheath flow.
A laser beam is irradiated onto the sample solution flow (sample flow) in the sheath flow, and the scattered light and fluorescence (green fluorescence, red fluorescence, etc.) emitted from each particle are analyzed. The particles to be analyzed range from several hundred to several hundred thousand, and cluster classification is performed by drawing particle size distributions and scatter diagrams of the scattered light intensity, green fluorescence, and red fluorescence. This technology is called a flow cytometer. This technology is applied to clinical testing devices that count and analyze blood cells in blood and red blood cells, white blood cells, epithelial cells, etc. in urine. However, this technology has difficulty in distinguishing between cell aggregates and particles with low concentrations. Therefore, a device equipped with a function of capturing still images of particles in a flow cytometer is desired. The present invention provides a particle analyzer that is inexpensive and has high image quality, and is equipped with a flow cytometer and a function of irradiating light for imaging on specific particles based on the conditions of scattered light and fluorescence after passing through a laser, and capturing an image.

In order to accurately measure the scattered light and fluorescence of minute particles and to ensure the analytical capacity, the flow cytometer requires a thin and fast sample flow. In reality, the sample flow flows at a speed of several meters to several tens of meters per second. In order to capture images of particles and cells in this fast-flowing sample flow, a light source with an emission time in nanosecond units is required. Generally, a pulsed laser is used. A pulsed laser has a short emission time of several nanoseconds to several tens of nanoseconds, high light intensity, and can capture still images of particles and cells flowing at high speed. However, laser light is highly coherent, which causes interference fringes and makes it difficult to obtain a clean image.

Patent Document 1 discloses an invention for imaging particles by reducing the coherence of pulsed laser light. In an embodiment of this invention, an optical fiber is used to reduce the coherence by passing the pulsed laser light through the optical fiber, thereby reducing the occurrence of interference fringes. However, this invention requires that the laser light be precisely incident on the optical fiber. Furthermore, the light emitted from the optical fiber must be shaped to illuminate uniformly, which has the disadvantage that the image becomes dark. In addition, since laser light is monochromatic, multiple lasers and optical fibers are required to capture color images.

In Patent Document 2 , an incoherent light source such as a xenon flash lamp is used instead of a pulse laser to capture an image. A light source with a relatively long light emission time is used, and as a device for this, an image intensifier with a gate function that can respond quickly is placed in front of the camera to shorten the exposure time, brighten the image, and obtain a clear still image without interference fringes. However, the image intensifier is expensive, and in order to capture an image in color, it is shown that multiple image intensifiers and cameras are installed by branching the light, which is complicated and expensive.

In addition, Non-Patent Document 1 discloses a product that uses a TDI camera (Time Delay Integration Camera) developed for the purpose of imaging moving objects. This can create still image data by irradiating a sample solution flowing through a flow cell with a continuous wave laser light, receiving the scattered light and fluorescence emitted by particles as they cross the laser light with an objective lens, and forming an image on the light receiving sensor of the TDI camera. Furthermore, the image is analyzed to calculate the scattered light intensity and fluorescent intensity of the particles, and a two-dimensional scattergram is created. However, the TDI camera is expensive, and the filters used for spectroscopy are complex and expensive.

Furthermore, Patent Documents 3 and 4 disclose inventions in which the flow rate is slowed down after measurement with a flow cytometer to capture images. This method allows images to be captured using a flash lamp because the flow rate is slowed down to capture images. However, it is necessary to slow down the flow rate sufficiently to capture images using a xenon flash lamp, which has a light emission time in the order of microseconds, which means that the flow path of the flow cell becomes large and long, and the flow cell holder becomes complicated.

The present invention relates to a flow cytometer that irradiates a sample flow of a sheath flow with a laser light and collects and analyzes scattered light and fluorescence from cells and particles, and a particle analyzer that selectively images cells and particles that meet specific conditions based on the results of the analysis. The present invention uses an incoherent LED as the light source for imaging. Furthermore, the pulse emission time of the LED is characterized by a half-width of 10 to 100 nsec.

LEDs are generally used to emit continuous light or pulse light with durations ranging from a few milliseconds to a few hundred milliseconds to prevent malfunctions or color changes due to heat generation, and therefore are not used to image cells flowing through a flow cytometer.

The LED used as the light source for imaging in the present invention can obtain high light intensity and a short emission time in nanosecond units by instantaneously passing electricity stored in a capacitor at several hundred volts to the LED using a switching transistor. In this way, high light intensity and short emission time can be obtained by charging a capacitor and instantaneously passing an electric charge, and the LED can be used as a stable light source without being damaged (Non-Patent Document 2).

Now, for example, if a red blood cell with a diameter of 7 μm (micrometers) in a sample flow flowing at 5 m (meters) per second is imaged using a xenon flash lamp with an emission time of 1.0 μsec (microseconds), a clear still image of the 7 μm (micrometers) red blood cell will not be obtained because the image will vibrate by 5 μm (micrometers) during the emission time. However, if the LED is pulsed with an emission time of 80 nsec (nanoseconds) or less, a clear still image can be captured with only a vibration of 0.4 μm (micrometers). The image vibration here is the distance L that the particle moves during the emission time, and can be calculated as emission time t x particle speed s = moving distance L.

Here, the light emission time of the LED pulse emission is defined and calculated as twice the half-width of the peak value of the pulse emission waveform (time and intensity). Therefore, a light emission time of 80 nsec results in a half-width of 40 nsec.

The shorter the light emission time of the LED light source used for imaging, the less vibration and the clearer the image obtained. However, as the light emission time becomes shorter, the capacity of the capacitor that stores the electric charge becomes smaller, the light emission intensity becomes weaker, and the image that can be captured becomes darker. Therefore, the lower limit of the pulse light emission time is set to 20 nsec (half width 10 nsec), which results in a vibration of 0.4 μm even at a relatively fast flow rate of 20 m per second. Furthermore, the upper limit of 200 nsec (half width 100 nsec) is the lower limit range of commercially available xenon flash lamps. If the light emission time exceeds the half width of 100 nsec, a xenon flash lamp may be used.

In the present invention, by making the optical axis of the laser light source for the flow cytometer and the optical axis of the light source for imaging perpendicular to each other, the lens systems of each light source and the objective lens system can be arranged close to each other, thereby shortening the flow path of the flow cell.

The sample flow path of the flow cell used in the present invention has a square hole with one side measuring 100 to several hundred microns in the area where scattered light and fluorescence are collected and analyzed, and if a flow cell that gradually expands in one direction from a position several millimeters to several tens of millimeters downstream from this area is used, the sample solution containing particles will expand laterally and become thinner as it flows while maintaining laminar flow. Using this flow cell, the focal position of the particles in the sample is stable, allowing for clear images to be taken.

In the present invention, the objective lens for receiving side scattered light and fluorescent light of the flow cytometer and the objective lens for imaging are the same, which simplifies the optical detection section of the particle analyzer.

In the present invention, the following effects can be obtained by using an LED that emits pulsed light in nanosecond units as the light source for imaging.
(1) LEDs emit incoherent light and can capture images with fewer interference fringes.
(2) By using surface-mounted LEDs, multiple LEDs can be arranged in a small area and made to emit light, thereby increasing the luminance intensity.
(3) Particle images can be easily captured as monochrome or color images. For monochrome images, a monochromatic LED is used and a monochrome camera is adopted.
For color images, a color camera is used, using white LEDs or three-color LEDs (RGB). Furthermore, if an epi-illumination system is used for the illumination system, a fluorescent image can be captured at the same time as a transmitted image. (4) Because LEDs are small, the illumination system for imaging can be made small, and the optical axis of the laser light source for the flow cytometer and the optical axis of the light source for imaging can be arranged perpendicularly, and the lens systems of each light source and the objective lens system can be arranged close to each other, shortening the flow path of the flow cell, making it possible to make the optical detection unit smaller and reduce costs.

1 is a block diagram showing a configuration of a first embodiment of the present invention; 2 is a circuit diagram of a power supply that causes an LED 21 of the present invention to emit light. FIG. 2 is a configuration diagram of a Kohler illumination including an LED 21 and an illumination system 22 according to the first embodiment. FIG. 2 is a diagram illustrating an example of the configuration of an illumination system other than the Kohler illumination system of the first embodiment. FIG. 4 is a diagram for explaining the timing of each signal in the particle analyzer of the present invention. FIG. 11 is a block diagram showing a configuration of a second embodiment of the present invention. FIG. 11 is a top view of a flow cell 8 according to a second embodiment of the present invention. FIG. 11 is a front view of a flow cell 8 according to a second embodiment of the present invention. FIG. 11 is a right side view of the flow cell 8 according to the second embodiment of the present invention. FIG. 11 is a bottom view of the flow cell 8 according to the second embodiment of the present invention. FIG. 11 is a block diagram showing a configuration of a third embodiment of the present invention. 13 shows the spectral transmittance characteristics of a dichroic mirror 61 used in the third embodiment of the present invention. 13 is an emission spectrum of a light source LED 21 used in the third embodiment of the present invention. FIG. 11 is a block diagram showing a configuration of a fourth embodiment of the present invention. 13 is an emission spectrum of a light source LED 64 used in the fourth embodiment of the present invention. 13 shows the transmission characteristics of the dichroic mirror 62 used in the fourth embodiment of the present invention. FIG. 11 is a schematic diagram of a cell passing through laser light in embodiment 4 of the present invention.

MODE FOR CARRYING OUT THEINVENTION

[Embodiment 1]
The following describes an embodiment of the present invention with reference to FIGS. 1 to 5. FIG.

FIG. 1 is a block diagram showing the flow of signals and image data, which is composed of an optical detection unit 100, a signal processing unit 31, and a microcomputer 33 of a particle analyzer.

The optical detection section 100 of this particle analyzer is composed of a flow cytometer (particle detection section), an imaging system (imaging section), and a fluid section. The particle detection section is composed of a flow cell 4, a semiconductor laser 1, a condenser lens 2 for focusing the laser light, a condenser lens 5 for collecting forward scattered light, a photodiode 6 for receiving the forward scattered light, an objective lens 11 for collecting side scattered light and fluorescence, dichroic filters 12, 13, and 14 for dispersing the side scattered light and fluorescence, and light receiving elements 15, 16, and 17 for receiving the side scattered light and fluorescence. The imaging section is composed of an LED 21 as a light source, an illumination system 22, a mirror 23, the flow cell 4, an objective lens 24 for imaging, and a digital camera 25. The fluid section includes a sample nozzle 3 and a fluid system for flowing a sample solution 36 and a sheath liquid 35, although they are omitted in the figure.

The sample solution 36 containing particles is diluted or stained in advance. The sample solution may be a biological sample containing a plurality of types of cells of different sizes, such as urine, blood, body cavity fluid, cervical tissue, etc., or may be artificial particles.

The sample solution 36 is discharged from the sample nozzle 3 into the flow cell 4, and at the same time, the sheath liquid 35 narrows the flow of the sample solution 36 to form a sheath flow. The thin flow of the sample solution (sample flow) 36 is irradiated with a laser beam emitted from a semiconductor laser 1, which is narrowed by a condenser lens 2. The semiconductor laser 1 oscillates continuously and uses wavelengths of 405 nm (nanometers), 488 nm, or 633 nm. The wavelength of 488 nm is the same as that of an argon ion laser, and the wavelength of 633 nm is the same as that of a HeNe laser, and is the most commonly used wavelength in flow cytometers. When cells or particles in the sample flow pass through this laser beam, forward scattered light, side scattered light, and fluorescence are generated. The scattered light generated in the same direction as the laser beam is called forward scattered light and reflects the size information of the particles. The side scattered light is generated in the perpendicular direction of the laser beam and reflects the internal information of the nucleus, granules, etc. in the cell. The fluorescence reflects the degree of staining in the cell and is received on the side where there is less stray light of the laser beam.

The forward scattered light is collected by the collecting lens 5, received by the photodiode 6, converted into an electric signal 41, and sent to the signal processing device 31. The side scattered light and fluorescence generated in a direction perpendicular to the laser light are collected by the objective lens 11, dispersed into side scattered light, green fluorescence, red fluorescence, etc. by the dichroic filters 12, 13, and 14, received by the light receiving elements 15, 16, and 17, converted into an electric signal, and sent to the signal processing device 31. The light receiving elements 15, 16, and 17 use highly sensitive photomultiplier tubes or avalanche photodiodes.

The forward scattered light signal 41 and side scattered light/fluorescence signal 42 sent to the signal processing device 31 are judged as to whether or not to image the particle based on a preset criterion. When a particle judged to be imaged reaches the imaging area, an LED light emission trigger signal 43 is sent to the trigger signal input terminal 115 of the LED power supply circuit to cause pulsed light emission, and at the same time, the particle is imaged by the digital camera 25. This digital camera 25 is a CCD camera or a CMOS camera. If it is desired to increase the number of images captured, a CMOS camera with a high frame rate is used. Furthermore, the digital camera 25 is of a global shutter type that can capture images at any timing. Illumination is performed by the illumination system 22 so that the particles are uniformly illuminated with the pulsed light emitted by the LED 21.

When capturing a monochrome image, it is inexpensive and has high image quality if a single-color LED is used for the LED 21 and a monochrome camera is used for the digital camera 25. The reason why monochrome images have high image quality is that they are less affected by chromatic aberration and have high pixel resolution. When capturing a color image, a white LED or three LEDs of red, green, and blue are used for the LED 21 and a color camera is used for the digital camera 25.

2 shows a power supply circuit including a light source LED 21. The LED 21 is used for high output with visible light or infrared light. A pulse drive charge is charged in a capacitor 111 from a high voltage DC power supply 113 through an electrical resistor 112. Then, an LED light emission trigger signal 43 is input to a trigger signal input terminal 115, which causes a switching transistor 114 to become conductive, through which the charge stored in the capacitor 111 is discharged. This discharge allows the LED 21 to emit high-luminance pulsed light with a light emission time measured in nanoseconds.

FIG. 3 is a structural diagram of an illumination device including a light source LED 21 and an illumination system 22. The illumination device constitutes a Kohler illumination. Kohler illumination is a method used for illumination of a microscope, and can provide uniform illumination. The configuration is an LED 21, a collector lens 52, a filter 53, a field stop 54, a window lens 55, an aperture stop 56, and a condenser lens 57. The illumination intensity can be adjusted by the aperture diameter of the aperture stop 56, and the area irradiated on the particle can be adjusted by the field stop 54. Furthermore, the wavelength band to be illuminated can be changed by the filter 53. Changing the wavelength band means changing the color in a color image. In this figure, the LED 21 is drawn in the commonly known shot-put type for the purpose of explanation, but a surface-mount type may also be used. In addition, multiple surface-mount types may be used to increase the brightness.

In this invention, the illumination device is explained as a high-performance Kohler illumination. However, to reduce costs, it may be composed of an LED 21 and a lens 51 as shown in Fig. 4. The light emitted by the LED 21 is collimated by the lens 51 and irradiated onto the particles.

The control of the timing of turning on or pulling up the LED light emission trigger signal 46 and the timing of putting the digital camera 25 into an exposure state will be described with reference to FIG. 5. From the forward scattered light signal 41 and the side scattered/fluorescent light signal 42 obtained from the particle detection unit, the end 44 of the particle signal of the forward scattered light or the side scattered/fluorescent light is detected, and it is determined that an image is to be captured during the time Tm1, and a camera shutter trigger signal 46 is sent to the digital camera 25, causing the digital camera to be in an exposure state. Next, when a particle reaches the imaging area located downstream (upper side in the figure) with respect to the semiconductor laser light 1, an LED light emission trigger signal 43 is sent to cause the LED 21 to emit light. The pulse emission time is 10 to 100 nanoseconds or less with a half-width, and a particle image is formed on the sensor of the digital camera. The captured image data is sent to the microcomputer 33, where it is stored and image processed.

[Embodiment 2]
A second embodiment of the present invention will be described with reference to FIGS.

As shown in FIG. 5, a flow cell 8 is used instead of the flow cell 4 of the first embodiment. The diagrams of the flow cell 8 are shown in FIGS. 7 to 10. The flow cell 8 is composed of a cell part 9 and a chamber part 10. The inner surfaces of the cell part 9 and the chamber part 10 are smoothly joined so that the sheath flow is formed without disturbance. A flow cytometer system that collects and analyzes scattered light and fluorescence has a square hole with a side of 100 to several hundred microns, and if a hole that gradually spreads in one direction from a position several millimeters to several tens of millimeters downstream from the laser irradiation position is used, the sample solution containing particles will spread laterally and become thinner as it flows while maintaining laminar flow. This stabilizes the focal position of the particles in the sample, allowing a clear image to be captured.

[Embodiment 3]
A third embodiment of the present invention will be described with reference to FIGS.

As shown in FIG. 11, the embodiment 1 for capturing a transmission image of a particle is added with a function of capturing a fluorescence image. The illumination method for capturing a fluorescence image forms an epi-illumination method with little stray light. The LED 21 of the light source uses a blue wavelength band that is dominant as an excitation light for generating fluorescence, as shown in FIG. 12. The illumination light (excitation light) emitted from the LED 21 passes through the mirror 23, the dichroic mirror, and the objective lens 63 and is irradiated onto the sample solution 36 flowing through the flow cell 4. The fluorescence emitted from the particle is again collected by the objective lens 63, and only the wavelength band of the fluorescence is reflected by the dichroic mirror 61, and an image is formed on the CMOS sensor of the digital camera 26 to capture the fluorescence image. FIG. 13 shows the transmission characteristics of the dichroic mirror 61. In this graph, the transmittance decreases from the long wavelength side near 500 nm, and instead the reflectance increases. The spectral characteristics of the LED 21 and the dichroic mirror 61 used here are only examples and are not limited to these.

[Embodiment 4]
A fourth embodiment of the present invention will be described with reference to FIG. 1 and FIG. 14 to FIG.

The difference from the first embodiment is that the condenser lens 5 for condensing the side scattered light and the fluorescent light and the objective lens 11 for capturing an image in the first embodiment (FIG. 1) are integrated into an objective lens 63 shown in FIG.

The conditions for realizing the embodiment are explained with reference to Fig. 14. The emission wavelength of the LED 64 in Fig. 9 has a peak at a wavelength of 700 nm or more as shown in the graph of Fig. 15 so that scattered light and fluorescent light do not become stray light when received. Next, the transmittance of the dichroic mirror 62 decreases from a wavelength of about 700 nm as shown in the graph of Fig. 16, and reflects only the light of the LED 64. Furthermore, the entrance window (filter) and objective lens 63 of the digital camera 25 have a high transmittance up to a wavelength of about 800 nm.

Fig. 17 is a schematic diagram of cells in the sample solution flow 36 flowing through the flow sul 4 immediately after passing through the laser light. If an objective lens 63 with a sufficiently wide field of view (dotted line) is adopted as shown in Fig. 12, the condenser lens that receives the scattered light and fluorescent light and the objective lens that captures the image can be integrated.

1: semiconductor laser, 2: condenser lens, 3: sample nozzle, 4: flow cell, 5: condensing lens, 6: photodiode, 8: flow cell, 9: cell section, 10: chamber section, 11: objective lens, 12, 13, 14: dichroic filter, 15, 16, 17: light receiving element, 21: LED, 22: illumination system, 23: mirror, 24: objective lens, 25: digital camera, 26: digital camera, 31: signal processing device, 32: display section, 33: microcomputer, 35: sheath liquid, 36: sample solution flow (sample flow), 41: forward scattered light signal , 42: side scattering/fluorescence signal, 43: LED light emission trigger signal, 44: end of particle signal, 46: camera shutter trigger signal, 51: lens, 52: collector lens, 53: filter, 54: field stop, 55: window lens, 56: aperture stop, 57: condenser lens, 61: dichroic mirror, 62: dichroic mirror, 63: objective lens, 64: LED, 100: optical detection unit, 111: capacitor, 112: electrical resistance, 113: high voltage DC power supply, 114: switching transistor, 115: trigger signal input terminal.

Claims (5)

a fluid means for forming a sheath flow in a flow cell together with a sample liquid containing test particles and a sheath liquid;
a laser light irradiation means for irradiating a test particle flowing through the flow cell with a laser light;
a photodetector that receives scattered light and fluorescent light emitted from the test particle;
A particle detection unit comprising:
a signal processing device that determines whether or not imaging is possible based on the intensity and time characteristics of the light received by the photodetector;
an imaging means for imaging a still image of the test particle determined to be imaged downstream of the particle detection unit with a digital camera by using an LED as a light source to emit pulsed light with a half-value width of 10 to 100 nanoseconds or less ;
A particle analyzer comprising an image processing unit for processing an image of a captured test particle. 2. The particle analysis device according to claim 1 , wherein a flow velocity in a region of the imaging means in the flow cell is configured to be slower than a flow velocity through the particle detection section. 3. The particle analyzer according to claim 2 , further comprising a flow path portion in which an inner diameter of the flow cell increases toward the downstream of the imaging portion, and the increase in the inner diameter increases gradually only in a direction perpendicular to the optical axis direction of the light source. 2. The particle analyzer according to claim 1 , which is configured to be able to capture a fluorescent image of the test particles. 2. The particle analyzer according to claim 1 , wherein the objective lens for the side scattered light and the fluorescent light of the light detecting section is the same as the objective lens for imaging.