JP4163301B2 - Scanning cytometer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a scanning cytometer that measures a cell by two-dimensionally scanning a specimen with a laser beam.
[0002]
[Prior art]
The cell population on the slide glass is scanned with a spot focused by a laser (laser beam), the fluorescence emitted by individual cells of the cell population is detected, and the scanned image data is processed to extract individual cell data. Japanese Patent Application Laid-Open No. 3-255365 discloses a “method and apparatus for measuring a plurality of optical characteristics of a biological specimen” (“Laser Scanning Cytometer (LSC)”).
[0003]
A laser scanning cytometer (hereinafter referred to as a “scanning cytometer”) (LSC) is a method for measuring a cell population of an isolated cell suspension sample with respect to a fixed laser and a spot on which the laser beam is focused. Unlike a flow cytometer that measures each cell by dividing it into several tens of thousands of water droplets per second by ultrasonic vibration and flowing it over a laser spot as a jet water stream, the cell population placed on a slide glass On the other hand, there is a difference in the basic configuration in that each cell is measured by optically and mechanically scanning the laser spot. However, a scanning cytometer (LSC) irradiates and excites a laser spot to a cell population biochemically labeled with a fluorescent dye, and measures the fluorescence emitted by each cell at high speed. The flow cytometer is the same as the flow cytometer in that the measurement result is presented as statistical data representing the immunological characteristics, genetic characteristics, cell proliferation, etc. of the population.
[0004]
Further, the scanning cytometer (LSC) records the coordinate position of each cell on the slide glass and can reproduce the coordinate position by the scanning stage. Thereby, individual cells showing photometric values corresponding to a specific subset of the statistical data can be moved into the microscope field and observed with a microscope. That is, the cytometer (LSC) is superior to the flow cytometer in that the microscope observation image can be associated with the cell data. The function of observing the microscopic morphology by sequentially reconstructing the position of individual cells under the microscope after measuring the cell population specimen is hereinafter referred to as “recall observation function” of a scanning cytometer (LSC).
[0005]
A typical medical biological research application of a scanning cytometer (LSC) is cell cycle analysis of cancer tumors. In cancer tumors, the property of “proliferation” is particularly important, but this does not give any knowledge even when microscopically observing each cell, and the hundreds to thousands of cells that make up the tumor tissue. Among the cells, it can be objectively expressed as a statistical numerical index by the ratio of the number of cells belonging to the stable phase of the cell division cycle, the DNA synthesis phase, and the division phase. Scanning cytometers (LSCs) provide such statistical numerical indicators, while specifying cell data belonging to a specific phase within the cell division cycle (eg, metaphase) to bring individual cells into the microscopic field. With the recall observation function, which is called sequentially, morphological examination can be performed under a microscope.
[0006]
On the other hand, a confocal laser scanning microscope (hereinafter referred to as “CLSM: Conforcal Laser Scanning Microscope”) has recently become widespread as an apparatus for observing a fluorescently labeled cell structure. The CLSM is capable of observing the optical cut surface at a high resolution compared to the conventional fluorescence microscope, acquiring the moving means in the optical axis (Z) direction and the optical cut image data string in the optical axis (Z) direction. In combination with image processing means for constructing and displaying a three-dimensional image, the cell structure can be observed three-dimensionally and analyzed.
[0007]
In the case of analysis of the cell cycle by the above-mentioned scanning cytometer (LSC), it is an important use to analyze the behavior of chromosomes in the middle, late and late phases especially at the cell division stage. Sharing with the recall observation function is expected. Further, from the viewpoint of the apparatus configuration technology, functional items such as laser irradiation, two-dimensional scanning, fluorescence detection, and scanning image data capture are common between the scanning cytometer (LSC) and the CLSM. It can be said that.
[0008]
On the other hand, the specifications of the common function items of the scanning cytometer (LSC) and the CLSM differ depending on the purpose of the apparatus and the performance to be achieved. Conventionally, the functions of the scanning cytometer (LSC) and the CLSM are different. No device with a function is provided.
[0009]
If the user needs sharing, the same scanning stage as the scanning cytometer (LSC) is installed in the CLSM and recalled as a system separate from the scanning cytometer (LSC). By using CLSM for observation, there is a possibility of realization as a combination of two system products.
[0010]
However, the user must own and use two system configuration units such as a microscope, a scanning stage, a laser light source, a scanning device, a fluorescence detector, and an image capturing computer, and there is a cost burden such as purchase cost and maintenance cost. There is a drawback that the space occupied by the apparatus in a laboratory or the like increases. In addition, the user must operate the two devices separately, resulting in inconvenience that the user experience is poor.
[0011]
For this reason, the scanning cytometer (LSC) and CLSM are mostly used in spite of the advantages of sharing the scanning cytometer (LSC) and CLSM in the medical and biological applications described above. There is no actual situation.
[0012]
[Problems to be solved by the invention]
Although there is no prior art that shares the use of the scanning cytometer (LSC) and CLSM, the technical problems will be described based on the prior applications of the scanning cytometer (LSC) and CLSM.
[0013]
Japanese Patent Laid-Open No. 3-255365 shows the idea of a scanning cytometer (LSC) as described at the beginning. Here, the spot size of the laser that irradiates the sample and scans two-dimensionally is assumed to be “equivalent to the cell size (within the same order of magnitude)”, but the fluorescence detection system is described as both a confocal system and a non-confocal system. Not. Regarding the spot size, an explanation is given for scanning the cell population specimen at a higher speed.
[0014]
On the other hand, according to Japanese Patent Laid-Open No. 7-209187, in order to acquire a “high resolution image” for morphological observation at the same time as scanning for photometry, it is sized as a confocal detection aperture (“aperture arranged at the imaging position”). Two or more different aperture diameters are prepared. According to the present invention, since a “high-resolution image” for morphological observation is acquired in advance at the same time as photometry, the captured “high-resolution image” is not reproduced during the recall observation of the scanning cytometer (LSC). Just call so Good.
[0015]
However, storing “high-resolution images” of thousands to tens of thousands of cell populations as data for each cell measurement of each cell population specimen requires enormous data storage capacity. For this reason, the system scale increases and the cost increases, and at the same time, the data storage / recall time becomes longer, and the throughput of the system decreases.
[0016]
In addition, when acquiring a “high resolution image”, the optical depth of focus becomes shallow. Therefore, when acquiring a high resolution image, it is necessary to perform focusing repeatedly in order to correct the focus shift during sample scanning. Disappear.
[0017]
Further, each cell structure is three-dimensionally combined with a moving means in the optical axis (Z) direction and an image processing means for acquiring an optically cut image data sequence in the optical axis (Z) direction to construct and display a three-dimensional image. When taking advantage of the features of CLSM that can be observed and analyzed morphologically, more time and data storage capacity are required to acquire the “high resolution image” sequence.
[0018]
In addition, the “high resolution image” that can be achieved here is because the spot size is relatively large (“equivalent to the cell size”) while irradiating a laser spot for photometry, and only the aperture on the fluorescence detection side is made small. It is impossible to achieve the CLSM theoretical resolution limit in both the in-plane direction and the optical axis direction. In other words, the “high resolution image” obtained in Japanese Patent Laid-Open No. 7-209187 is unsatisfactory as CLSM in terms of resolution and optical cutting performance, and fulfills the purpose of the above-mentioned common use of the scanning cytometer (LSC) and CLSM. There is a disadvantage that can not be.
An object of the present invention is to provide a scanning cytometer having a CLSM function while suppressing an increase in user equipment cost and an increase in occupied area.
[0019]
[Means for Solving the Problems]
A scanning cytometer according to the present invention includes a laser light source that emits laser light, an objective lens that focuses the laser light from the laser light source on a specimen, and the laser light source to the pupil of the objective lens. As a parallel beam The beam diameter of the incident laser beam To fill that pupil By spreading, the sample is irradiated with laser light having a spot diameter corresponding to the diffraction limit size determined by the numerical aperture of the objective lens. A first state and a second state in which measurement as a cytometer is performed by making the beam diameter of laser light incident as a parallel beam into the pupil of the objective lens sufficiently smaller than the pupil. Luminous flux diameter switching means switchable to, Whether in the first state or the second state Scanning means for scanning the specimen with laser light from the laser light source; optical path switching means capable of switching light from the specimen to an optical path of a confocal detector and an optical path for non-confocal detection; and A detector for detecting light, and the beam diameter switching means irradiates the laser beam with a spot diameter corresponding to a diffraction limit size. First state When switching to, the optical path of the confocal detector is selected.
Also The present invention provides a laser light source that emits laser light, an objective lens that converges the laser light from the laser light source onto a specimen, and the laser light source to the pupil of the objective lens. As a parallel beam By switching the beam diameter of the incident laser light, The luminous flux diameter fills the pupil A first state in which a spot diameter focused on the specimen corresponds to a diffraction limit size determined by a numerical aperture of an objective lens; The diameter of the light beam is sufficiently smaller than the pupil and is condensed on the specimen. Luminous flux switching means switchable to a second state equivalent to the cell size; Whether in the first state or the second state A scanning means for scanning the specimen with laser light from the laser light source and a detector for detecting light from the specimen, and switching means for confocal detection of light from the specimen and means for non-confocal detection are switched. In the first state, the optical cross-sectional image is acquired by switching to the confocal detection means, and in the second state, the cell measurement is performed by switching to the non-confocal detection means. This is a characteristic scanning cell measuring apparatus.
Also The present invention includes a laser light source that emits laser light, an objective lens that focuses the laser light from the laser light source on a specimen, and the laser light source to the pupil of the objective lens. As a parallel beam The beam diameter of the incident laser beam To fill that pupil By spreading, the sample is irradiated with a laser beam having a spot diameter corresponding to the diffraction limit size determined by the numerical aperture of the objective lens. A first state and a second state in which measurement as a cytometer is performed by making the beam diameter of laser light incident as a parallel beam into the pupil of the objective lens sufficiently smaller than the pupil. Luminous flux diameter switching means switchable to, Whether in the first state or the second state A scanning means for scanning the specimen with laser light from the laser light source and a detector for detecting light from the specimen, and switching means for confocal detection of light from the specimen and means for non-confocal detection are switched. The laser beam is irradiated with a spot diameter corresponding to the diffraction limit size. In the first state Sometimes switching to the confocal detection means to acquire an optical cross-sectional image and measure with a scanning cytometer Switched to the second state In this case, the scanning cytometer is switched to the non-confocal detection means.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
The configuration of a scanning cytometer according to an embodiment of the present invention is shown in FIG.
The scanning cytometer (LSC) 10 has a CLSM function, and can selectively perform either LSC measurement or CLSM measurement.
[0021]
The apparatus 10 includes an excitation light introducing unit 100 that emits excitation light, a dichroic mirror 12 that separates excitation light and fluorescence, an optical deflector 20 that two-dimensionally scans excitation light, a pupil projection lens 32, The objective lens 34 and the fluorescence detection unit 200 are included.
[0022]
Each of the excitation light introducing unit 100 and the fluorescence detecting unit 200 includes two systems of an optical system for LSC measurement and an optical system for CLSM measurement.
The excitation light introducing unit 100 includes a laser light source 102 that emits a laser beam as excitation light, and a spot projection lens 104 for LSC measurement.
[0023]
Further, for CLSM, the excitation light introducing unit 100 includes two convex lenses 118 and 120 constituting a beam expander, and four optical path bending mirrors 106, 114, and 116 for allowing light to pass through the beam expander. And 110 are provided.
[0024]
The two optical path bending mirrors 106 and 110 are selectively detachably disposed on the optical path between the laser light source 102 and the spot projection lens 104, and between the spot projection lens 104 and the dichroic mirror 12, respectively. In other words, the two optical path bending mirrors 106 and 110 are inserted into the optical path as indicated by the solid line, depending on the type of measurement, by the slide mechanisms 108 and 112 (light beam diameter switching means), respectively, It is comprised so that it may remove | deviate from an optical path as shown by.
[0025]
The optical deflector 20 includes two galvanometer mirrors whose driving directions are different by 90 °, and is configured to be able to deflect light beams in XY directions orthogonal to each other by changing the directions of the respective reflecting surfaces. Only one galvanometer mirror is shown in the figure for convenience of explanation.
[0026]
The fluorescence detection unit 200 includes a collector lens 204 for LSC measurement and a photodetector for detecting fluorescence, such as a photomultiplier tube 202.
For CLSM, the fluorescence detection unit 200 converts the convex lens 218 that condenses the fluorescence, the confocal pinhole 222 disposed at the focal position thereof, and the fluorescence that has passed through the confocal pinhole 222 into a parallel light beam. A convex lens 220 and four optical path bending mirrors 206, 214, 216, and 210 for guiding the fluorescence to the confocal pinhole 222 and guiding the passing light to the photomultiplier tube 202 are provided.
[0027]
The two optical path bending mirrors 206 and 210 are selectively detachably disposed on the optical path between the dichroic mirror 12 and the collector lens 204 and between the collector lens 204 and the photomultiplier tube 202, respectively. In other words, the two optical path bending mirrors 206 and 210 are inserted into the optical path as indicated by the solid line or are imaginary by the slide mechanisms 208 and 212 (optical path switching means), depending on the type of measurement. It is configured to be removed from the optical path as shown.
[0028]
The apparatus 10 further includes a stage 50 on which the specimen 40 is placed, a controller 60 that controls each part, an external input device 62 for inputting necessary information, and a monitor 64 for displaying measurement results and the like. Have.
[0029]
The stage 50 includes two tables that can move in directions orthogonal to each other, and the placed specimen 40 can be moved in XY directions orthogonal to each other.
The controller 60 controls the optical deflector 20, the stage 50, and the four slide mechanisms 108, 112, 208, and 212, processes measurement data obtained from the photomultiplier tube 202, and monitors processing results and necessary information. 64.
[0030]
In the LSC measurement, the four optical path bending mirrors 106, 110, 206, and 210 are moved to positions indicated by imaginary lines by the four slide mechanisms 108, 112, 208, and 212, respectively, and are removed from the optical path.
[0031]
The laser emitted from the laser light source 102 is reflected by the dichroic mirror 12 through the spot projection lens 104, enters the objective lens 34 through the optical deflector 20 and the pupil projection lens 32, and is condensed on the surface of the specimen 40. The
[0032]
The laser emitted from the laser light source 102 is a substantially parallel light beam, but strictly speaking, it proceeds while gradually spreading from the light beam in the laser tube. The spot projection lens 104 is a weak convex lens that suppresses this spread, and correctly forms a light beam in the laser tube on the surface of the specimen 40.
[0033]
Therefore, the laser is incident on the pupil position of the objective lens 34 with a light beam diameter sufficiently smaller than the aperture diameter of the pupil of the objective lens 34.
In order to scan a wide range in the LSC measurement, the optical deflector 20 drives only one galvanometer mirror 22, for example, the galvanometer mirror 22 that scans the light beam in the X direction. The stage 50 is moved in a direction (Y direction) orthogonal to the scanning direction.
[0034]
In this way, by combining the movement of the spot limited to a narrow range by the optical deflector 20 and the movement of the specimen 40 by the stage 50 at a low speed but a wide range, the spot formed on the specimen surface is on the specimen. Scanned over a wide range.
[0035]
From sample 40 This The fluorescent light collected by the objective lens 34 passes through the pupil projection lens 32 and the optical deflector 20, then passes through the dichroic mirror 12, passes through the collector lens 204, and enters the head-on photomultiplier tube 202. It is photoelectrically converted.
[0036]
The fluorescence from the optical deflector 20 toward the photomultiplier 202 is a substantially parallel light beam, but strictly speaking, it is slightly diverged when the specimen is defocused in the direction approaching the objective lens. The collector lens 204 is a convex lens for making it converge, and the light receiving surface of the photomultiplier tube 202 is connected to the optical deflector 20 in a substantially imaging relationship. Since the pupil plane of the objective lens 34 and the optical deflector 20 are connected to the imaging relationship by the pupil projection lens 32, the pupil plane of the objective lens 34 and the light receiving surface of the photomultiplier tube 202 are connected to the imaging relationship. Yes. As a result, the photometric quantity does not easily change even when the light quantity distribution on the light receiving surface is defocused due to the scanning by the optical deflector 20 or the variation in the thickness or position of the specimen by pupil plane photometry.
[0037]
Since the fluorescence from the specimen 40 is emitted from the entire surface of the pupil aperture of the objective lens 34, the aperture diameter of the collector lens 204 is the maximum defocus diameter of the objective lens 34 to be used and the worst defocus when the specimen 40 approaches the objective lens. It is determined in consideration of the conditions.
[0038]
Photoelectron Increase The output of the double tube 202 is input to the controller 60. The controller 60 is an optoelectronic Increase Scanning image data based on the photoelectric signal from the double tube 202 is processed to quantitatively measure the amount of intracellular components of each cell in the sample. The controller 60 further records the coordinate position data of each cell in the specimen, statistically analyzes the photometric value of each individual cell, and based on the coordinate position data of each individual cell belonging to a specific subset, The position of the specimen 40 is controlled by 50, and a desired cell is rearranged in the microscope visual field, so that the apparatus 10 can realize a recall observation function.
[0039]
In the CLSM measurement, the four optical path bending mirrors 106, 110, 206, and 210 are moved to positions indicated by solid lines by the four slide mechanisms 108, 112, 208, and 212, respectively, and are arranged on the optical path.
[0040]
The laser emitted from the laser light source 102 is reflected by the two optical path bending mirrors 106 and 114, passes through the beam expander composed of the two convex lenses 118 and 120 having different focal lengths, and then the two optical path bending mirrors 116 and After being reflected by 110, it is reflected by the dichroic mirror 12, enters the objective lens 34 through the optical deflector 20 and the pupil projection lens 32, and is collected on the specimen surface 40.
[0041]
The laser beam is magnified by a beam expander at a magnification that determines the beam diameter by the focal length of the convex lens 120 / the focal length of the convex lens 118. As a result, the laser is incident on the pupil position of the objective lens 34 with a light beam diameter larger than the aperture diameter of the pupil of the objective lens 34.
[0042]
In the CLSM measurement, since the spot is moved with high position accuracy, scanning with the spot on the specimen 40 is performed using two galvanometer mirrors of the optical deflector 20 having high speed and high position controllability.
[0043]
From sample 40 This The fluorescent light collected by the objective lens 34 passes through the pupil projection lens 32 and the optical deflector 20, passes through the dichroic mirror 12, is reflected by the two optical path folding mirrors 206 and 214, and then collected by the convex lens 218. The light is shining and connects the confocal spots. The light that has passed through the confocal pinhole 222 is approximately collimated by the convex lens 220, reflected by the two optical path bending mirrors 216 and 210, and then incident on the head-on type photomultiplier tube 202 and subjected to photoelectric conversion. .
[0044]
The convex lens 218 is designed to form a confocal spot having a diffraction limit diameter. Further, the confocal pinhole 222 is configured such that the controller 60 can adjust the position and select an opening size by a turret (not shown) so that the confocal pinhole 222 is accurately arranged in conformity with the confocal spot size. It is preferable.
[0045]
In general, the CLSM measurement is performed following the LSC measurement. For example, after grasping the overall tendency of a sample by LSC measurement, CLSM measurement is generally performed on a portion that attracts the user's attention.
[0046]
As described above, since the coordinate position data of each cell in the specimen is already acquired at the time of the LSC measurement, in the CLSM measurement, the controller 60 observes information input from the external input device 62, for example, the user observes. Depending on the desired location, the apparatus 10 moves the stage 50 and places specific cells in the specimen 40 on the optical axis.
[0047]
The stage 50 preferably has a function of moving the specimen 40 along the optical axis direction, that is, the Z direction, so that the apparatus 10 can acquire an optical cross-sectional image utilizing the features of the confocal optical system. . Furthermore, the apparatus 10 can construct a three-dimensional image by acquiring the optically cut image data sequence while controlling the position of the specimen 40 in the Z direction by the controller 60.
[0048]
As can be seen from the above description, the apparatus 10 controls the position of the optical path bending mirrors 106, 110, 206, and 210 inside the excitation light introducing unit 100 and the fluorescence detection unit 200, thereby scanning the cytometer (LSC). ) And CLSM functions can be switched.
[0049]
This switching is preferably performed by preparing a dedicated GUI (Graphical User Interface) for the external input device 62, controlling the slide mechanisms 108, 112, 208, and 212 by the controller 60, and controlling the optical deflector 20 and the stage 50. It is done by doing. As a result, the user can switch between the scanning cytometer (LSC) and the CLSM without any particular awareness.
[0050]
Therefore, high-resolution three-dimensional morphology observation by CLSM can be applied during recall observation with a scanning cytometer (LSC).
In the present embodiment, an apparatus having one laser light source 102 and one photomultiplier tube 202 has been described as an example. However, of course, the present invention can be applied to a sample that is multiple-stained with a plurality of fluorescent reagents. In order to simultaneously measure a plurality of different fluorescences, the present invention can be applied to an apparatus having one or a plurality of laser light sources and a plurality of photomultiplier tubes.
[0051]
The present invention is not limited to the embodiments described above, and includes all implementations that are performed without departing from the scope of the invention. For example, even when one laser light source is used as in the present embodiment, the optical path system is switched in addition to switching the optical path and switching the light beam diameter in each optical path, for example, on the optical path of the laser light source. It is possible to switch the light beam diameter by moving the lens on the optical path via a lens that can perform afocal zoom, and multiple lenses with different focal lengths, for example, on the slide mechanism Setting Therefore, the beam diameter can be switched by switching the lens having a desired focal length in the beam diameter expanding optical path used in the present embodiment.
[0052]
The invention includes implementations illustrated in the following sections.
1. A laser beam is focused on a cell population sample by a microscope objective lens and scanned two-dimensionally. Fluorescence from a cell excited by the laser is detected by a detector, and a scan formed by a photoelectric signal detected by the detector. Image data is taken into a computer, the amount of intracellular components of each cell in the cell population specimen is quantitatively measured by image processing on the scanned image data, and the coordinate position data of each cell in the cell population specimen is recorded. Scanning type that statistically analyzes the photometric values of the cells of each cell, and controls the stage based on the coordinate position data of individual cells belonging to a specific subset to move each cell within the microscope field of view In the cytometer, it is equivalent to the diffraction limit size determined by the numerical aperture of the objective lens. Spot diameter laser light Means for irradiating a specimen with two-dimensional scanning and The fluorescence generated by this two-dimensional scan It has a means to detect confocal fluorescence.
2. The apparatus according to claim 1, wherein the objective is lens Numerical aperture so Equivalent to the diffraction limit size to be determined Spot diameter laser light Means for irradiating a specimen with two-dimensional scanning and The fluorescence generated by this two-dimensional scan Of the means for detecting confocal fluorescence, at least one component is at least partially shared with the scanning cytometer.
3. 2. The apparatus according to claim 2, further comprising image processing means for acquiring a moving means in the optical axis (Z) direction and an optically cut image data sequence in the optical axis (Z) direction to construct and display a three-dimensional image. It is characterized by that.
4). In the apparatus according to item 2, the diffraction limit size is determined by the numerical aperture of the objective lens. Spot diameter laser light Among the means for irradiating a specimen with two-dimensional scanning, at least a laser and an optical deflector are provided with the scanning Measurement It is shared with the apparatus, and includes a laser beam diameter conversion means.
5. 2. The apparatus according to claim 2, wherein at least the fluorescence detector of the means for detecting confocal fluorescence is shared with the scanning cytometer, and at least of the fluorescence focusing lens switching means and the confocal pinhole insertion / extraction switching means. It is characterized by having one side.
6). In the apparatus according to item 2, the diffraction limit size is determined by the numerical aperture of the objective lens. Spot diameter laser light Means for irradiating a specimen with two-dimensional scanning and The fluorescence generated by this two-dimensional scan The means for detecting confocal fluorescence comprises a multi-pinhole disk scanner and an image sensor.
[0053]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the scanning cytometer which has the function of CLSM is provided, suppressing the economical burden of a user, and the increase in an occupation area. Thereby, CLSM can be applied at the time of recall observation of a scanning cytometer (LSC), and more preferable form observation can be performed.
[Brief description of the drawings]
FIG. 1 shows a configuration of a scanning cytometer according to an embodiment of the present invention.
[Explanation of symbols]
20 Optical deflector
34 Objective lens
100 Excitation light introduction part
118 Convex lens
120 Convex lens
200 Fluorescence detector
202 photoelectrons Increase Double pipe
218 Convex lens
222 Confocal pinhole

Claims (3)

A laser light source for emitting laser light;
An objective lens for focusing the laser light from the laser light source on the specimen;
A laser beam having a spot diameter corresponding to the diffraction limit size determined by the numerical aperture of the objective lens by expanding the beam diameter of the laser beam incident as a parallel beam from the laser light source to the pupil of the objective lens so as to fill the pupil. Is switched between a first state in which the specimen is irradiated and a second state in which the diameter of the laser beam incident as a parallel beam on the pupil of the objective lens is made sufficiently smaller than the pupil to perform measurement as a cytometer. Possible beam diameter switching means;
Scanning means for scanning the specimen with laser light from the laser light source regardless of whether the first state or the second state ;
An optical path switching means capable of switching light from the specimen to an optical path of a confocal detector and an optical path for non-confocal detection;
A detector for detecting light from the specimen, and the optical path of the confocal detector when the laser beam is switched to the first state in which the laser beam is irradiated with a spot diameter corresponding to the diffraction limit size by the beam diameter switching means. A scanning cytometer characterized by the selection. A laser light source for emitting laser light;
An objective lens for converging the laser light from the laser light source on the specimen;
By switching the light beam diameter of the laser light incident as a parallel beam from the laser light source to the pupil of the objective lens, the spot diameter that the light beam diameter fills the pupil and condenses on the specimen is the numerical aperture of the objective lens. Switching between the first state corresponding to the determined diffraction limit size and the second state where the beam diameter is sufficiently smaller than the pupil and the spot diameter focused on the specimen is equal to the cell size. Possible luminous flux switching means;
Scanning means for scanning the specimen with laser light from the laser light source regardless of whether the first state or the second state ;
A detector for detecting light from the specimen,
The means for detecting confocal light and the means for detecting non-confocal light are switched between the specimens, and in the first state, the means is switched to the means for detecting confocals to obtain an optical cross-sectional image, and the second In this state, the scanning cell measuring apparatus is characterized in that the cell measurement is performed by switching to the non-confocal detection means. A laser light source for emitting laser light;
An objective lens for focusing the laser light from the laser light source on the specimen;
A laser beam having a spot diameter corresponding to the diffraction limit size determined by the numerical aperture of the objective lens by expanding the beam diameter of the laser beam incident as a parallel beam from the laser light source to the pupil of the objective lens so as to fill the pupil. Is switched between a first state in which the specimen is irradiated and a second state in which the diameter of the laser beam incident as a parallel beam on the pupil of the objective lens is made sufficiently smaller than the pupil to perform measurement as a cytometer. Possible beam diameter switching means;
Scanning means for scanning a specimen with laser light from the laser light source regardless of whether the first state or the second state ;
A detector for detecting light from the specimen,
The means for confocal detection of light from the specimen and the means for non-confocal detection are switched,
Get the optical cross-sectional image by switching the unit at times the confocal detection of the first state of irradiating the laser beam at the diffraction limit equivalent to the size of the spot diameter, to a second state for measuring the scanning cytometer scanning cytometer when switched, characterized in that the switching to a means for detecting the non-confocal.

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