JPH0616037B2 - Automated cytology imaging device - Google Patents

Description

TECHNICAL FIELD The present invention relates to an apparatus for observing cells and the like under a microscope and temporarily accumulating a specimen image thereof. For example, cells from the accumulated specimen images can be used. The present invention relates to an automated cytodiagnosis apparatus that provides effective images for so-called cytodiagnosis automation that automatically classifies types of cells.

[Prior Art] As a conventional device equipped with a cell image capturing means, there is an automated system for cytodiagnosis described in “Clinical Examination” vol.28 No.3, issued March 1984, pp.292-297. . For example,
The cell image capturing means of the "automatic detection system for cancer cells" shown in Fig. 1 on page 293 of the same document projects the light of the lamp condensed by the condenser lens onto the prepared and fixed preparation specimen, and the transmitted light of the specimen. Captured with a microscope and photographed with a color TV camera. The image that becomes an electrical signal is A / D converted and stored in the frame memory in the computer. These images are subjected to image processing using a computer, and cancer cells and the like are extracted and classified.

Another conventional technique is a so-called flow cytometer described in "Flow cytometry handbook", pages 24 to 25, issued on November 30, 1984. As shown in Figure 1 on page 25 of the same document, this device arranges a flow cell in the observation section under the microscope, sends the cell suspension into the sheath flow at high speed, and irradiates it with laser light to irradiate each cell. The scattered light and fluorescence of are collected by a microscope and the amount of light is measured by a phototodetector and stored in a memory. Since a large amount of information about these cells can be obtained in a short time, the computer mainly performs statistical processing.

[Problems to be Solved by the Invention] Among the above-mentioned conventional techniques, the former example is to fix a cell as a preparation in a microscope observation section and simultaneously image a plurality of cells. The number of cells in the field of view of the field varies, which adversely affects comparison between samples, and the number of cells in the field of view is also limited, so that the number of cells in the statistical processing is limited. There were problems such as few things. Further, in the case of performing image processing by a computer, the misalignment of cells or the overlapping of cells may cause a reduction in processing time.

Since the flow cytometer, which is the latter example of the above-mentioned conventional technique, uses the sheath flow technique, a large amount of cells can be sent to the measurement point in a short time, which is advantageous for statistical processing. However, there is a problem in that the present method cannot detect a small number of cells that exist at a frequency of only one in thousands.

It is an object of the present invention to provide an apparatus for taking a large number of cells and images of cells that are separated from each other and aligned.

[Means for Solving the Problems] The automated cytodiagnosis apparatus of the present invention has an introduction-side flow path end and a discharge-side flow path end in the outer peripheral portion and the central portion, respectively, and cells and the like can be aligned in a line. A spiral capillary flow path formed in the same plane with a tube diameter of a certain degree, and a cell suspension and a sheath fluid are introduced to wrap the cell suspension in the sheath fluid to contract the suspension. A sheath flow cell provided with a sheath flow forming part for aligning cells in a suspension liquid in a row and flowing out to the introduction side flow path end of the spiral capillary flow path, and projecting light onto the sheath flow cell. A light source, a microscope that captures an image of the entire spiral capillary channel of the sheath flow cell, a photographing unit that captures the image captured by the microscope, and a storage unit that stores the captured image. Must be combined with an imaging device Is characterized by.

[Operation] The sheath flow cell is a device capable of wrapping a cell suspension in a sheath liquid to cause a contraction, and flowing cells one by one in a capillary channel. In the present invention, this capillary channel is extended and converged in a spiral shape, so that cells flow in a spiral shape in a line in the channel until reaching the end. In this state, light is projected from the light source over the entire spiral flow path, and light from each cell due to the light is collected by a microscope, captured by a photographing means on the optical axis extension, and accumulated by a storage image means.

As described above, since the long capillary channel is made into a spiral shape to be compact, many cells can be captured and photographed in the same visual field of the microscope. In this photographing, each cell is photographed in a state of being separated one by one by the sheath flow cell.

It should be noted that when each cell is analyzed one by one from the accumulated image, it is possible to perform high-speed processing by searching along the spiral shape.

[Embodiment] An embodiment of the present invention will be described below with reference to the drawings of FIGS. 1 to 4.

The present embodiment comprises a cell imaging device (Fig. 1) and an image processing device (Fig. 2) that analyzes the captured image. FIG. 3 shows a configuration diagram of a sheath flow cell having a spiral capillary flow path used in the cell imaging device, and FIG. 4 shows a state after imaging of a disk film which is an image storage means also used in the cell imaging device. It is a figure.

The cell imaging apparatus shown in FIG. 1 will be described. For a sheath flow cell 6 for observing cells in a sheath flow, a white strobe light source 1 is provided below the sheath flow cell 6, and the light is condensed and applied to the sheath flow cell 6. There is a condenser lens 2 for this, and a microscope 3 for collecting the light transmitted through the spiral flow path of the sheath flow cell 6 and a camera 4 for taking an image of the collected light on the optical axis extension are provided on the upper side. There is. A signal cable 13 for transmitting the flow rate in the seal flow cell 6 is connected from the sheath flow cell 6 to the control unit 5. A signal cable 14 for transmitting a light emission timing is connected from the control unit 5 to the white strobe light source 1, and a signal cable 15 for transmitting a shutter opening / closing signal is connected to the camera 4. In the sheath flow cell 6, a tube 10 for taking in the cell suspension 7, a tube 11 for taking in the sheath liquid 8 and a tube 12 for discharging the drainage 9 are provided.
Is provided.

As shown in FIG. 3, the tube 10 for cell suspension is connected to the sample liquid nozzle in the center of the sheath flow forming part 18, and the tube 11 for sheath liquid is connected to the sheath liquid port 23. A spiral capillary flow path 19 is provided downstream of the seal flow forming portion 18, and the flow path end 20 is connected to the drainage pipe 12. Conduits 22 and 21 extend from the sheath liquid port 23 and the vicinity of the flow path end 20, respectively, and are guided to different surfaces of the diaphragm type differential pressure gauge 16. From the differential pressure gauge, a signal cable 13 for transmitting a flow rate signal to the control unit 5 is output. The flow path in the sheath flow cell 6 is made of a glass plate, a stainless plate, or a silicon plate, and transparent plates such as glass are attached to the upper and lower surfaces of the plate so that light can pass therethrough. .

FIG. 4 shows a state after the image recording disc film 24 built in the camera 4 is photographed, and an image 25 of cells flowing in the spiral capillary channel 19 is recorded as shown in the figure.

The image processing apparatus shown in FIG.
A white light source 27 that irradiates transmitted light to the light source and a condenser lens 28 that collects the light are provided below the disc film 24. Further, on the upper side, there is a television camera 29 at a position where the transmitted light can be collected, and the television camera 29 and the image processing unit 31 are connected by a signal cable. A display television 32 is connected to the image processing unit 31. The disc film 24 is supported so as to be rotatable around the center of the circle as shown by an arrow 26. The arrow 30 indicates the sliding direction of the television camera.

The operation of the present embodiment having the above configuration will be described below.

In the sheath flow cell 6 having the spiral flow path shown in FIG. 3, the cell suspension 7 is from the tube 10 and the sheath liquid 8 is from the tube 1.
Introduced from 1. In the sheath flow forming section 18, the cell suspension 7 discharged from the central nozzle is wrapped with the sheath liquid 8 to be contracted, and the cells 17 are aligned in an example as shown in the drawing and guided to the spiral capillary channel 19. After reaching the flow path end 20, the cells 17 are discharged through the drainage pipe 12. The flow rate of the fluid passing through the spiral capillary channel 19 depends on the sheath liquid port 23 and the channel end 20.
It can be known by measuring the differential pressure between the two. The principle of this is described in the technical data “Fluid measurement method” edited by the Japan Society of Mechanical Engineers, page 214, “Laminar flow meter”. In this embodiment, the respective pressures of the sheath liquid port 23 and the flow path end 20 are introduced to both sides of the diaphragm type differential pressure gauge 16 to which the strain sensors using the conduits 22 and 21 are attached, and the differential pressure is expressed by the strain amount of the diaphragm. To detect. Detection signal is cable 13
Is transmitted to the control unit 15.

Since the upper and lower surfaces of the sheath flow cell 6 are covered with a transparent body, the cells 17 in the flow path can be observed from the upper and lower surfaces of the spiral capillary flow path 19. Letting ΔT be the time it takes for the leading end of the cell suspension 7 to reach the flow path end 20 after starting to flow in a sheath flow, ΔT can be found by dividing the length of the spiral capillary flow path 19 by the flow velocity. . In the control unit 5 shown in FIG. 1, the above ΔT is calculated using the capillary channel length stored in advance and the flow rate detection signal transmitted from the cable 13 at any time. The time for filling the end of the capillary channel 19 to the end 20 is predicted, and at that moment, a switch signal is sent to the white strobe light source 1 and the camera 4 through the signal cables 14 and 15. The strobe light emitted from the white strobe light source 1 in response to this signal is condensed by the condenser 2 and illuminates the capillary channel 19 from the lower surface of the sheath flow cell 6. The strobe light transmitted through the flow path 19 is collected by the microscope 3 and guided to the camera 4. In the camera 4, the shutter is opened by the switch signal previously received from the control unit 5, and the transmitted light is imaged on the built-in photographic film surface.

In this embodiment, a disc-shaped film 24 as shown in FIG. 4 is used so that the spiral capillary channel 19 can be photographed without omission. In the image of the spiral capillaries photographed on the disc film 24, the cell images 25 aligned as shown in FIG. 4 are recorded at high density.

The disc film 24 on which the cell images photographed as described above are recorded is applied to the image processing apparatus shown in FIG. The disc film 24 is rotated symmetrically with respect to its center point as indicated by arrow 26. The light from the white light source 27 is condensed on the disc film 24 by the condenser lens 28. The television camera 29 that captures the transmitted light from the film 24 is designed to slide in the radial direction of the disc film 24 together with the irradiation optical system as shown by the arrow 30, and the rotation speed and the sliding speed of the disc film 24 are adjusted appropriately. By doing so, the cell image can be collected without fail along the spiral capillary channel.

The cell image image thus obtained is sent to the image processing unit 31 and subjected to various image processing. The results are output to the display TV 32 or the like.

The spiral capillary flow path 19 of the sheath flow cell 6 shown in this embodiment has, for example, a flow path width of 50 μm and a space between the flow paths of 100.
μm, the total length of the channel is 50 mm, and the outer diameter of the spiral channel is 3 mm. In order to actually manufacture such a sheath flow cell, it is effective to process a flow path in a thin plate (stainless steel plate, silicon plate, glass plate or the like) by using a fine processing technique such as etching, and stack and assemble them.

In order to capture the entire spiral capillary channel 19 having an outer diameter of 3 mm without impairing the resolution of each cell image, a wide-field objective lens may be used. If the channel width, the distance between the channels, and the like are made smaller, the denseness of the channels is increased, and the outer diameter of the spiral capillary channel 19 can be reduced accordingly, and the objective with a narrow field of view and high magnification can be obtained. A lens can be used, and a higher-definition cell image can be obtained. Such ultra-fine processing can be easily performed by using a fine processing technique such as etching.

Consider, for example, blood as a cell suspension to be passed through the sheath flow cell 6 having the above dimensions. The number of red blood cells in human blood is about 5 × 10 ⁶ cells / mm ³ in adults. This is diluted about 2 times, and the sheath liquid is flowed at a flow rate of about 10 times that. Then, a sheath flow having a diameter of about 20 μm is formed, and erythrocytes flow in the position of 10 μm on average in the sheath flow. Since the length of the capillary flow path 19 of this sheath flow cell is 50 mm, about 500 red blood cells are added to the capillary flow path 1 at any time.
9 is satisfied. Therefore, in this example, it is possible to capture an image of about 500 aligned cells in one shot.
It is said that the number of cells to be processed by the cell image diagnostic equipment using a preparation specimen as described in the section of the prior art is limited to about 100 cells, while on the other hand, the number of cells processed by the conventional flow cytometric cell diagnostic equipment is 1000 to several cells. Thousands are processed. The device shown in this example has an intermediate performance between these in terms of the number of cells to be processed, but if a single sample is divided and photographed repeatedly, a population equivalent to that of a flow cytometer can be created. You can Moreover, since images of individual cells are accumulated and image diagnosis is possible, the number of cases in which rare cells are missed can be significantly reduced. In this way, this device can be said to be a device that combines the features of a conventional cytodiagnostic device using image processing and a flow cytometer.

A characteristic feature of this embodiment is that the cell image photographing device shown in FIG. 1 and the image processing device shown in FIG. 2 can be separated by interposing the disc film 24. This is convenient when going out of the hospital and doing an outpatient examination. The cell imager has a simple structure, and the entire device can be made small enough to be portable, so specimens taken from patients on a business trip can be converted into detailed image information on the spot. Eliminates the need to store and take samples back. For example, one of the cytodiagnosis is a urine sediment for examining the contained cells in urine. This is a test in which the urine of a person who was found to have occult blood in the test urine due to the reaction of the test paper at the first examination is re-collected at a later date to identify cells by a microscope or the like. In this case, the result of the initial examination may not match the result of the urinary sediment, and it is considered not good to shift the examination date and time even in the case of tension. According to the cell image capturing apparatus of the present embodiment, a sample requiring urine sediment can be imaged on the spot, brought back, and subjected to image processing, so that the above problem can be easily solved.

In this embodiment, a white strobe light source is used as the irradiation light source, but monochromatic light such as laser light may be applied to a sample that has undergone pretreatment such as dyeing with a fluorescent dye to capture the fluorescence amount.

It is also possible to make a holography of each cell by the scattered light and the reference light from the cells in the spiral capillary channel, record it as a hologram, and analyze the stereoscopic image later.

In this embodiment, a disk film is used to increase the processing efficiency of the image processing apparatus, but of course, a commercially available winding film may be used. In this case, the image processing apparatus may trace the irradiation light and the photographing camera unit along the spiral shape of the flow path.

A video tape may be used as a means for accumulating an image, or an image may be once accumulated in a frame memory and a signal may be passed from there to the image processing section. Storage media such as magnetic disks, floppy disks, and optical disks can of course be used.

FIG. 5 shows another embodiment of the present invention. The same components as those of the above-described embodiment are represented by the same numbers, and detailed illustration and description thereof will be omitted. In this embodiment, the function of a flow cytometer is combined with the cell image capturing apparatus of the above embodiment. A half mirror 35 is inserted between the white strobe light source 1 and the condenser lens 2 shown in FIG. Similarly, a half mirror 36 is inserted between the microscope 3 and the camera 4. The laser light 34 of the laser light source 33 is the half mirror 3
It is reflected at 5, and is irradiated onto the cells 17 at the inlet of the spiral capillary channel of the sheath flow cell 6 through the condenser lens 2. Light 41 from cell 17 is half mirror 3
Reflected at 6, the photodetector 37 is installed at a place where the amount of light is measured. The control section 38 is connected to the photo detector 37 and the laser light source 33 in addition to the white strobe light source 1 and the camera 4 by signal cables 39 and 40, respectively.

The present embodiment having the above configuration operates as follows.

In the above-described embodiment, the time ΔT required for the cells 17 to enter the spiral capillary flow channel 19 to the end of the flow channel is calculated based on the measurement of the flow rate, and imaging is performed when this time ΔT has elapsed. On the other hand, in the present embodiment, imaging is performed when the passage of a fixed number of cells is detected by laser photometry. In addition, in the present example, the size of each cell, or the amount of fluorescence of cells pretreated with a monoclonal antibody or fluorescent staining is also measured. The laser irradiation point is a spiral capillary channel 1
The light information from each cell is captured by the photodetector 37 and is sent to the control unit 38 by the signal cable 3
Sent through 9. The control unit 38 accumulates these pieces of information and, upon confirming that a fixed number of cells have passed, sends a command to the laser light source 33 via the signal cable 40 to temporarily stop light emission. Immediately after that, an image capturing command is given to the white strobe light source 1 camera 4, and an image of cells flowing through the sheath flow cell is captured.

By monitoring the fixed number of cells reflected on the disc film 24 with a laser light counter as in the present embodiment, different samples (for example, different dilution ratios or cells flowing at unequal intervals) may be detected. If), the population can be kept stable. Furthermore, the greatest feature is that the information of each cell accumulated in the control section and each cell image taken are completely synchronized, and measurement information such as scattered light by laser light or fluorescence and morphological It is possible to know information (image information) at the same time and for a large number of cells. There has been an attempt to capture the passage image of cells by emitting a pulse lamp each time cells pass the laser irradiation point, but as the number of passing cells increases, the frequency of pulse lamp emission becomes too late, It was impossible to capture the cell image without omission by synchronizing with the optical information. The present embodiment can effectively solve these problems.

EFFECTS OF THE INVENTION According to the present invention, many cells can be captured in the same visual field of a microscope. In addition, it is possible to obtain an image in which individual cells are separated and aligned in a spiral. Therefore, compared with the conventional image processing apparatus for cytodiagnosis, image diagnosis can be performed on each cell at a higher speed, and statistical processing with the same number of cells as a flow cytometer is possible.

[Brief description of drawings]

FIG. 1 is a perspective view of a cell imaging apparatus according to an embodiment of the present invention, FIG. 2 is a perspective view of the same image processing apparatus, and FIG. 3 is a partial view for clarifying the internal structure of the sheath flow cell in the same embodiment. Fourth perspective view, broken away and shown in plan view,
FIG. 5 is a plan view showing a state after the photographing of the disc film for photographing used in the same embodiment, and FIG. 5 is a schematic configuration diagram of another embodiment of the present invention. 1 ... White strobe light source 2 ... Condenser lens, 3 ... Microscope 4 ... Camera 6 ... Sheath flow cell, 7 ... Cell suspension 8 ... Sheath liquid 18 ... Sheath flow forming part 19 ... Spiral shape Capillary flow path 24 ... disk film 33 ... laser light source, 37 ... photo detector

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hideaki Kambara 502 Jinritsucho, Tsuchiura-shi, Ibaraki Prefecture Hiritsu Seisakusho Co., Ltd.Mechanical Research Institute (72) Inventor Kuniyoshi Tsubouchi 502 Kintate-cho, Tsuchiura-shi, Ibaraki Hitsuritsu Co., Ltd. Machinery Research Laboratory (72) Inventor Hideo Enoki 502 Jinritsucho, Tsuchiura City, Ibaraki Prefecture Hitate Manufacturing Co., Ltd. (72) Inventor Muneo Kawamoto 4-6 Kanda Surugadai, Chiyoda-ku, Tokyo Hitachi Ltd. ( 56) References JP 54-21395 (JP, A) JP 61-247962 (JP, A) JP 60-24709 (JP, B2) JP 62-12461 (JP, B2)

Claims (1)

[Claims] 1. An outer peripheral portion and a central portion each have an inlet-side channel end and an outlet-side channel end, and a tube diameter such that cells and the like can be aligned in a line, and are formed in the same plane. Introducing the spirally-shaped capillary flow path and the cell suspension and the sheath liquid, wrapping the cell suspension with the sheath liquid to cause a contraction, and align the cells and the like in the suspension in a row to form the spiral. Sheath flow forming section for flowing out to the introduction side flow path end of the capillary flow path, a light source projecting onto the sheath flow cell, and an image of the entire spiral flow path of the sheath flow cell A microscope that captures
An imaging device comprising an imaging device for capturing an image captured by the microscope and an image storage device for accumulating the captured image, and a combination of the sheath flow cell and the imaging device. Automated cytology imaging equipment.