JPH06235691A - Imaging flow cytometer - Google Patents

Abstract

PURPOSE:To provide a flow image analyzer which can image and analyze a transmitted light image and a fluorescent image simultaneously and efficiently. CONSTITUTION:The imaging flow cytometer comprises a normal emission light source 45 for normally monitoring a cell passing through the imaging area of a cell imaging video camera, and a continuous emission exciting light source 35 for taking a fluorescent image of the cell. A flash lamp 1 is lighted when a line sensor 14 detects passage of a cell. When the cell is moved by a predetermined distance, an intensifier 22 is turned ON for a short time thus obtaining an intensified fluorescent still image. This constitution allows simultaneous imaging of a transmitted light image using the light of flash lamp 1 and a fluorescent image using the exciting light on one screen.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides a sheath flow of a sample solution containing particle components such as blood and urine, irradiating the sample solution stream with light to obtain a still image, and image processing the sample solution. The present invention relates to a device for performing analysis such as classification and counting of particle components, and more particularly to a particle image analyzer (imaging flow cytometer) adapted to capture a still image of particles by using continuous emission light. .

[0002]

2. Description of the Related Art It has been practiced in the past to perform analysis such as classification of particles by applying excitation light to stained particles such as cells and detecting fluorescence emitted from the particles. Flow cytometers and fluorescence microscopes are known.

A flow cytometer can detect the amount of fluorescence emitted from individual particles.

With a fluorescence microscope, a more detailed fluorescence image can be observed. Further, the obtained fluorescence image is subjected to image processing. Further, Japanese Patent Laid-Open No. 63-269875 discloses an image pickup device capable of capturing three types of light images of ultraviolet, visible and infrared.

On the other hand, an apparatus for picking up an image of particles flowed in a flow and analyzing the particles by image processing is disclosed in Japanese Patent Application Laid-Open No. 5-54
7-500995 and U.S. Pat. No. 4,338,024.
No.

Further, there is also known a device in which an image pickup area is constantly monitored, and it is detected that particles in a flow arrive at the area to efficiently pick up a particle image.

[0007]

In order to obtain a fluorescence still image of a sufficient amount of light for particles flowing at high speed, the conventional method is as follows.
A pulsed laser with a large amount of light (for example, an argon pulsed laser) was used for excitation. Such pulsed lasers are large and expensive. On the other hand, if an even cheaper light source (for example, a continuous emission argon laser) can be used, the device can be made compact and inexpensive as a result. An object of the present invention is to provide an imaging flow cytometer capable of obtaining a good fluorescence still image even when using a light source of constant light emission.

[0008]

In order to achieve the above object, the present invention is to obtain a fluorescence still image by using an image intensifier with a gate function, the constitution of which is a sample liquid containing a particle component to be detected. A flow cell in which a flow path for flowing is formed, a light source that irradiates the sample solution in the flow cell with light, an imaging unit that captures a still image of the particles to be detected in the sample solution irradiated by this light source, and In an imaging flow cytometer including processing means for performing desired analysis based on image data from the imaging means,
A first light source for irradiating the sample liquid in the flow cell with light, an image amplifying means for optically amplifying a particle image obtained by the first light source, and an image capturing the amplified particle image. A first image pickup means, a second light source for constantly irradiating the sample liquid in the flow cell with light, a second image pickup means for scanning the particle image formed by the second light source, and Control means for controlling the operation of each means, the first light source is a continuous light emission fluorescence excitation light source, the image by the first light source is a particle fluorescence image, and the control means is the second Particles are detected based on a signal from the image pickup means, and based on this detection, the image amplification means is operated during the image-capable period of the first image pickup means, whereby a fluorescence still image of the particles is picked up by the first image pickup means. Enabled imaging flow cytometer It is an.

[0009]

EXAMPLE FIG. 1 shows an example of the present invention. In the imaging flow cytometer of the present invention, in addition to the second light source (in the example, a near-infrared semiconductor laser) 45 for monitoring cell passage and the line sensor 14 which is the second imaging means, a fluorescent image is captured. An excitation light source (not a pulsed light source but a continuous light source) 35 which is a first light source, and an image intensifier 22 with a gate function for multiplying weak fluorescence are added. Furthermore, a video signal switcher (image signal selecting means) for combining the main parts of the transmitted light image and the fluorescent image to obtain a single screen is added.

The steps will be described below in order. A near-infrared semiconductor, which is a second light source, is provided for guiding the sample solution containing the fluorescently stained cells to the flat sheath flow cell 6 and constantly monitoring the passage of the cells through the imaging area of the video camera 24. The laser 45 constantly illuminates the imaging area. The light from the semiconductor laser 45 is collimated by the collimator lens 46, reflected by the dichroic mirror 4 through the cylindrical lens 47, and further narrowed by the condenser lens 5 perpendicularly to the cell advancing direction. The image is picked up on the imaging area of the line sensor 14 which is the original image sensor. In this example, the line sensor 14
The image pickup area A2 of the monochrome video camera 24A, which is the first image pickup means, is shown in FIG.
Also, it is provided slightly above the middle of the upper half (divided by a dotted line) of the image pickup area A3 of the color video camera 24B which is the third image pickup means. B2 is the second light source 4
This is a light irradiation region of 5.

Imaging area A by the light of the semiconductor laser 45
1, the transmitted light from A3 passes through the objective lens 8 and the dichroic mirror 4A to pass through the dichroic mirror 1
It is reflected by 2 and is imaged on the line sensor 14. From the line sensor 14, the scanning cycle time for one line (tens of μ
The voltage corresponding to the photoelectric conversion accumulated amount of each pixel exposed for (sec) is sequentially output. By performing signal processing on this voltage signal, if it is determined that the cell 7 has crossed the imaging area A2 of the line sensor 14 during the even field period of the video camera 24, the flash lamp light (white light) is emitted. The flash lamp 1 as the third light source emits light. For details, see JP-A-2-
185794, JP-A-2-185795, JP-A-4
Since it is described in Japanese Patent No. 270961, etc., its explanation is omitted.

The processing time from when the cell 7 crosses the imaging area A2 of the line sensor 14 to when the flash lamp 1 is triggered is 100 to 200 μse when the scanning period of the line sensor 14 is 50 μsec.
c and the flow velocity of cells in the flow cell 6 is 50 m
If it is m / sec, the cells move 5 to 10 μm during this period. Therefore, the transmitted light image of the cells obtained by irradiating the flash lamp light can be reliably captured in the upper half area of one image pickup screen A1, A3.

The light of the flash lamp is collimated by the collimator lens 9 and radiated through the dichroic mirror 4 and the condenser lens 5 almost uniformly over the entire image pickup area A3 of the video camera 24B. The transmitted light image of the cell is transmitted through the objective lens 8, the dichroic mirrors 4A and 12 and a portion through the beam splitter 11, and is imaged on the CCD area sensor of the video camera 24B which is the third imaging means.

At this time, the transmitted light image largely reflected by the beam splitter 11 is formed on the photocathodes of the filter 20 and the image intensifier 22. However, since the gate signal is turned off so that the image intensifier 22 does not operate, no image appears on the output surface of the image intensifier 22. The signal S2 for turning on the gate is generated by the cell passage determination / light source control unit 28.

When the cell 7 moves to the position of the lower half of the imaging area A1 (below the dotted line), the laser light from the first light source 35, which is a fluorescence excitation light source, is irradiated. This laser light is made horizontally long by the cylindrical lens 36,
The light is reflected by the dichroic mirror 4A, further narrowed down by the objective lens 8 in a slender and vertical direction with respect to the cell traveling direction, and is irradiated around the middle of the lower half of the imaging area A1 of the video camera 24A (see FIG. 2). ). At this time, the cells 7 are moving to this irradiation area B1. As described above, the first light source 35 continuously emits light.

When the cell 7 reaches the fluorescent excitation light irradiation area B1, the cell emits fluorescent light. This fluorescence is transmitted through the dichroic mirror 12 through the objective lens 8, is largely reflected by the beam splitter 11, is transmitted through the excitation light cut filter 20, and is transmitted to the image intensifier 2
An image is formed on the photoelectric surface of 2. At that time, the gate signal to the image intensifier 22 is turned on to operate for several μsec. This gate signal is generated by the cell passage determination / light source control unit 28. For example, the flow rate of the cells in the flow cell is 50 mm / sec, the video camera 24
When the image pickup area A1 of A is 150 × 150 μm, the image intensifier 2 is provided about 1.5 msec after the flash lamp 1 for transmitted light image pickup is irradiated.
It may be controlled so that the gate of 2 is turned on.
The operating time varies depending on the flow velocity of the cells, but is a time such that the fluorescence image does not largely shake. For example, when the flow velocity is 50 mm / sec, it may be set to 10 μsec or less.

When the gate signal is supplied to the image intensifier 22, a high voltage is applied to the MCP (micro channel plate) inside the image intensifier 22, and the weak fluorescent image formed on the photocathode is magnified tens of thousands of times. It is multiplied and output to the fluorescent surface. The image on the fluorescent surface is formed on the CCD of the monochrome camera 24A by the relay lens 18.

As described above, the image intensifier 2
Activating 2 for only a short time is not only for obtaining a static fluorescence image. The intense light reflected by the beam splitter 11 at the time of capturing the transmitted light image is formed on the photocathode of the image intensifier 22. At this point, the image intensifier 22 receives the gate signal S2 as shown in FIG. Since it is OFF, image amplification is not performed and the image intensifier 22 can be protected from excessive input. Also, the laser light from the second light source for cell passage monitoring is always imaged on the photocathode of the image intensifier 22. Normally, the gate signal is OFF, and the gate signal is ON for a short time when necessary for exposure. The effect can be minimized by shortening the time.

FIG. 3 is an example showing the timing of the flash lamp light irradiation after detecting the cells passing through the imaging area A2 and the timing of the gate signal S2 of the image intensifier 22, and signals for controlling these. Is produced by the cell passage determination / light source control unit 28 of FIG.
S1 is a particle detection signal, and S3 is a signal for flash lamp irradiation.

In the sequence as described above, as shown in FIG. 4, an image D3 in which the transmitted light image C3 of the cell is shown on the upper portion of the screen is obtained by the color video camera 24B, and the image of the cell is obtained by the monochrome video camera 24A. An image D1 in which the fluorescent image C1 is shown at the bottom of the screen is obtained. The image signals of these images D3 and D1 are sent to the video signal switcher 30 which is an image signal selecting means. The video signal switcher 30 switches the image signal. That is, when scanning the top of the image,
By selecting the image signal from the color video camera 24B and selecting the image signal from the monochrome video camera 24A when scanning the lower part of the screen, the image D
Image D4 in which the upper half of image 3 and the lower half of image D1 are combined
To get An image D4 obtained by combining the transmitted light image C3 of the cell and the fluorescent image C1 into one screen is sent to the image processing device 25,
Various types of image processing and image analysis are performed on the cells.

[0021]

[Function] Second light source (near infrared light source for particle monitoring) 45
And a first light source (flash lamp light source for exciting fluorescence)
All 35 always emit light. When the particles reach the imaging area A2 of the second imaging unit 14, the control unit 28 detects the particles based on the signal from the second imaging unit 14.
On the other hand, the particles emit fluorescence when they reach the irradiation area of the first light source 35. The fluorescent image of the particle is formed on the amplification means 22. Therefore, when the particles are detected, the control unit 28 operates the image amplification unit 22 for a short time to obtain an amplified fluorescence still image. This fluorescence still image is the first
The image is taken by the image pickup means 24A.

The image obtained by the first image pickup means 24A and the image obtained by the third image pickup means 24B are switched and combined during the scanning by the image signal selecting means 30, and a fluorescence image and a transmitted light image of the same particle are obtained. An image can be obtained as a single screen on which both of.

[0023]

[Effect] Since the arrival of particles is monitored and the particles are within the irradiation area of the fluorescence excitation light, the image amplification means is gated and operated only for a short time. Therefore, the fluorescence excitation light source is of the continuous emission type. Even if the light source is used and the amount of fluorescent light is small, a good fluorescent still image can be obtained. Since the continuous light emitting type light source is small and inexpensive, as a result, a small and inexpensive imaging flow cytometer can be realized.

Further, it is possible to collect a necessary part (a part where particles are reflected) from a plurality of images by using the image signal selecting means so as to collect one image.

[Brief description of drawings]

FIG. 1 shows the configuration of an imaging flow cytometer according to the present invention.

FIG. 2 is an explanatory diagram showing an example of a light irradiation area and an imaging area of a flow cell unit.

FIG. 3 shows a timing chart of light irradiation timing and an intensifier ON gate signal.

FIG. 4 shows an explanatory diagram of an image signal selecting means.

[Explanation of Codes] 6 ... Flow Cell, 1 ... Third Light Source, 45
... second light source, 35 ... first light source, 24A
... first imaging means, 14 ... second imaging means, 28
... control means, 24B ... third imaging means,
22 ... Image amplification means.

Claims (5)

[Claims] 1. A flow cell in which a flow path for flowing a sample solution containing a particle component to be detected is formed, a light source for irradiating the sample solution in the flow cell with light, and a sample in the sample solution irradiated by the light source. An image pickup means for picking up a still image of the detection particles;
A first light source (35) for irradiating the sample liquid in the flow cell (6) with light in an imaging flow cytometer including a processing means for performing a desired analysis based on image data from the imaging means. An image amplification means (22) for optically amplifying the particle image obtained by the first light source, a first imaging means (24A) for capturing the amplified particle image, and a flow cell A second light source (45) for constantly irradiating the sample solution with light, a second image pickup means (14) for scanning the particle image formed by the second light source (45), And a control means (28) for controlling the operation of each means, wherein the first light source (35) is a continuous light emission source for exciting fluorescence, and the image by the first light source is a particle fluorescence image. The control means (28) is the above-mentioned Of detecting the particles on the basis of a signal from the imaging means to operate the said image amplifying means during the imaging period of the first imaging means based on the detection, thereby,
An imaging flow cytometer, wherein a fluorescence still image of particles is taken by the first imaging means. 2. The imaging flow cytometer according to claim 1, further comprising a third light source (1) for irradiating the sample solution in the flow cell with light, and particles obtained by the third light source. Third image pickup means (24B) for picking up a still image, and image signal selection means (30) for selecting the first image signal of the first image pickup means and the third image signal of the third image pickup means. Is further provided, and the control means detects the particles to be detected based on the image pickup signal of the second image pickup means, and based on this detection, the first and
A particle image is picked up by the third image pickup, and the image signal selecting means (30) is switched during scanning of the image pickup screen, so that the particle image in the first image and the particle image in the third image are switched. An imaging flow cytometer characterized by combining the part in which is shown to obtain a single screen image. 3. The imaging flow cytometer according to claim 1, wherein the first imaging unit has a two-dimensional imaging region on the flow of the sample liquid, and the second imaging unit has the flow of the sample liquid. It has a line-shaped imaging region on the
An imaging flow cytometer, wherein the imaging area of the imaging means is formed so as to cross the flow of the sample liquid. 4. The imaging flow cytometer according to claim 2, wherein the first and third imaging means each have a two-dimensional imaging region on the flow of the sample liquid, and the second imaging means An imaging area on the line is provided on the flow of the sample liquid, and the imaging area of the second imaging means is the first or third imaging area.
An imaging flow cytometer, which is formed so as to cross the flow of the sample liquid in the imaging region of the imaging means. 5. The imaging flow cytometer according to claim 3 or 4, further comprising means for making the irradiation light from the second light source an ellipse.