JP3686713B2 - Scanning cell measuring device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a scanning cell measuring apparatus capable of scanning a cell population with a spot converged with a light beam and automatically inspecting micronuclei of cells.
[0002]
[Prior art]
A cell population on a slide glass is scanned with a spot focused with a light beam (laser beam), and fluorescence or the like emitted from individual cells of the cell population is detected and data processed. A measuring method and apparatus "(hereinafter referred to as a scanning cytometer) is disclosed in Japanese Patent Laid-Open No. 3-255365.
[0003]
In the scanning cytometer, the spot where the laser and the laser beam are focused is fixed, and the cell in the state of isolated suspended liquid flows on the spot as a jet water flow. There are differences in the basic configuration in that the laser spot is scanned optically and mechanically.
[0004]
However, a scanning cytometer irradiates and excites a laser spot onto a biological cell population biochemically labeled with a fluorescent dye, and measures the fluorescence emitted by each cell at high speed, and the measurement result is measured by the cell population. The flow cytometer is the same as the flow cytometer in that it is intended to be presented as statistical data representing the immunological characteristics, genetic characteristics, cell proliferation, and the like.
[0005]
Scanning cytometers have the following features compared to flow cytometers:
1. In addition to the amount of fluorescence, morphological information such as area and shape can be obtained by two-dimensional scanning of the laser spot.
[0006]
2. The coordinate position of each cell on the slide glass is recorded and the coordinate position can be reproduced, so that each cell presenting individual cell data in the statistical data is reproduced in the microscope field for microscopic observation. Images and cell data can be associated with each other.
[0007]
As a laser light source in a scanning cytometer, the blue wavelength (488 nm) of an air-cooled argon laser is usually used. In addition, as a fluorescent dye for labeling a specimen, FITC (Fluorescein is othiocyanate) emitting green-yellow fluorescence is generally used as a fluorescent labeling dye combined with a fluorescent photo antibody method, and as a fluorescent quantitative dye for intranuclear or intrachromosomal DNA. Is generally PI (Propidium Iodide) of amber fluorescence.
[0008]
In addition, there is a pigment called AO (Acridine Orange), which emits amber fluorescence when bound to RNA and emits green-yellow fluorescence when bound to DNA. AO emits two different colors of fluorescence at one excitation wavelength, and is used for two-parameter measurement of RNA and DNA in a flow cytometer.
[0009]
[Problems to be solved by the invention]
Recently, a mutagenicity test by a micronucleus test has been conducted as a safety test for drugs and foods. This mutagenicity test has become more and more important in recent years due to circumstances such as clarification of product liability.
[0010]
There are two types of micronucleus tests: cultured cells (in vitro) and laboratory animal blood (in vivo). The former is a basic experimental method, and the latter is more practical. Method.
[0011]
In the method using blood from experimental animals, a slide specimen smeared from bone marrow or peripheral blood is used, and several hundred to thousands of red blood cells are visually observed under a microscope, and the ratio of those in which the micronuclei are expressed in the red blood cells is examined.
[0012]
Micronucleus specimens are usually stained with AO (Acridine Orange) and observed with a fluorescence microscope. At this time, the whole red blood cell is confirmed by the amber fluorescence combined with the RNA component in the red blood cell (especially the young red blood cell), and the presence or absence of the micronucleus in the red blood cell is visually confirmed by the green-yellow fluorescence combined with the DNA component of the micronucleus. Confirm.
[0013]
In the micronucleus inspection by microscopic observation, 100 to 1000 red blood cells are observed with the naked eye while moving the stage manually, and the measurement time and the pain of the operator are extremely large.
[0014]
Theoretically, in order to improve measurement accuracy, it is necessary to increase the number of specimens, that is, the number of red blood cells to be measured, but in the measurement by microscopic observation, considering the measurement time and the pain of the operator, in reality, The number of measured cells cannot be increased significantly.
[0015]
Further, in the conventional visual observation, since the form is observed and judged by the human eye, the reliability as objective data is lacking. In addition, the fluorescence amount of micronuclei (that is, the amount of DNA) could not be quantified.
[0016]
In addition, the measurement work to select the red blood cells from the various blood cells and platelets mixed in the smear specimen to check for the presence of micronuclei depends on the micromorphology, and a flow cytometer where micromorphological information cannot be obtained is used. Can not.
[0017]
The present invention has been made in view of the above circumstances, and an object thereof is to provide a scanning cell measuring apparatus capable of automatically counting and measuring a fine structure of a cell or a component constituting the structure. To do.
[0018]
[Means for Solving the Problems]
  Accordingly, first, in order to achieve the above object, the invention according to claim 1 is a light beam for two-dimensionally scanning a light beam spot on a cell population stained and labeled with single or plural fluorescent dyes having different spectral characteristics. Scanning means and first fluorescence for detecting fluorescence that labels a minute structure in the cell or a component constituting the structure, from fluorescence excited by the light beam scanned by the light beam scanning means Detection means; second fluorescence detection means for detecting fluorescence that labels the entire cell out of fluorescence from the cells excited by the light beam scanned by the light beam scanning means; and the first fluorescence detection means For scanned images obtained by fluorescence detected byOf the light beamSpatial filtering means for performing spatial filtering to emphasize a spot, and a first image that calculates a first image area that exceeds a first threshold among scanning image areas obtained by fluorescence detected by the second fluorescence detection means An area calculation unit and a scanning image obtained by fluorescence detected by the first fluorescence detection unit within the first image area calculated by the first image region calculation unit by the spatial filtering unit A second image region that exceeds the second threshold is calculated from the scanned image regions that have been filtered.And calculating a second image region that exceeds the second threshold for the scanned image that is not filtered by the spatial filtering means.Second image area calculation meansDetecting the presence or absence of a minute structure in the cell or a component constituting the structure, which is equal to or less than that of the light beam, using a second image region based on the scanned image on which the spatial filtering has been performed, Quantitative measurement of the second image region is performed using the second image region based on the scanned image that is not filtered.This is a scanning cell measuring apparatus.
[0019]
  Further, the invention according to claim 2 comprises: a light beam scanning means for two-dimensionally scanning a light beam spot on a cell population stained and labeled with a single or a plurality of fluorescent dyes having different spectral characteristics; and the light beam scanning means First fluorescence detection means for detecting fluorescence from a minute fluorescence-labeled portion inside a cell nucleus or chromosome among fluorescence excited by a light beam scanned by the light beam, and the light beam scanning means Of the fluorescence from the cells excited by the scanned light beam, second fluorescence detection means for detecting fluorescence that labels the whole cell nucleus or chromosome, and fluorescence detected by the first fluorescence detection means For the resulting scanned imageOf the light beamSpatial filtering means for performing spatial filtering to emphasize a spot, and a first image that calculates a first image area that exceeds a first threshold among scanning image areas obtained by fluorescence detected by the second fluorescence detection means An area calculation unit and a scanning image obtained by fluorescence detected by the first fluorescence detection unit within the first image area calculated by the first image region calculation unit by the spatial filtering unit. A second image region that exceeds the second threshold is calculated from the scanned image regions that have been filtered.And calculating a second image region that exceeds the second threshold for the scanned image that is not filtered by the spatial filtering means.Second image area calculation meansDetecting the presence or absence of a minute structure in the cell or a component constituting the structure, which is equal to or less than that of the light beam, using a second image region based on the scanned image on which the spatial filtering has been performed, Quantitative measurement of the second image region is performed using the second image region based on the scanned image that is not filtered.This is a scanning cell measuring apparatus.
[0020]
  Furthermore, the invention according to claim 3In the invention according to claim 1 or 2, the size of the spatial filtering operator is a size that matches a spot of the same degree as a beam spot scanned on the specimen, and has a lower limit. The image processing apparatus further comprises storage means for storing the number of second image areas calculated by the second image area calculation means in the first image area calculated by one image area calculation means.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is a diagram showing a multi-parameter fluorescence photometric optical system of a scanning cell measuring apparatus according to an embodiment of the present invention.
[0025]
The light beam emitted from the laser 11 is reflected by the main dichroic mirror 12 and reflected / deflected by a galvanometer mirror 13 that rotates about a rotation axis (not shown) orthogonal to the paper surface.
[0026]
The light beam reflected and deflected by the galvanometer mirror 13 is imaged on the objective lens image 15 by the pupil projection lens 14 to form a scanning spot scanned in the left-right direction on the paper surface.
[0027]
The scanning spot is projected onto the specimen surface 17 by the objective lens 16, and scans the cells on the specimen as a minute spot. Fluorescence from the fluorescent label of individual cells excited by the microspots goes back to the galvanometer mirror 13 to the optical path.
[0028]
Here, since the galvanometer mirror 13 is coupled with the pupil of the objective lens 16 by the pupil projection lens 14, the fluorescence from the specimen does not depend on the scanning deflection angle of the galvanometer mirror 13, and the laser Go up the light path.
[0029]
The fluorescence from the specimen passes through the main dichroic mirror 12 and enters the sub dichroic mirror 18. Here, of the fluorescence from the cells, the short wavelength component (for example, greenish yellow) is reflected by the sub dichroic mirror 18 and enters the first photomultiplier tube 19 to be detected photoelectrically. Of the fluorescence from the cell, a long wavelength component (for example, amber color) is transmitted through the sub-dichroic mirror 18 and incident on the second photomultiplier tube 20 to be detected photoelectrically.
[0030]
Thereby, two fluorescent components can be detected simultaneously by the first photomultiplier tube 19 and the second photomultiplier tube 20. Further, the sample surface can be scanned two-dimensionally with a laser spot by moving the sample 17 in the direction orthogonal to the paper surface on the scanning stage while scanning the laser in the left-right direction with the optical deflection means by the galvanometer mirror 13, Two-component scanning photometry using a combination of two different fluorescent dye labels spectroscopically becomes possible.
[0031]
FIG. 2 is a diagram showing the configuration of the electrical system of the scanning cell measuring apparatus according to the same embodiment.
Photoelectric signals detected by two photomultiplier tubes (PMT) 21a and 21b are processed by a signal processing system provided for each detection signal (referred to as a channel), and a memory board on an expansion slot in the computer 31 32 is transferred to and stored in a memory circuit for each channel provided in 32.
[0032]
That is, the respective photoelectric signals detected by the two photomultiplier tubes (PMT) 21a and 21b are amplified by the head amplifiers (Head Amps) 22a and 22b, and then analog integrated via the mixers 23a and 23b. The noise is removed by analog (Analog Integ.) 24a and 24b, and converted into 12-bit digital signals by analog / digital converters (A / D) 25a and 25b.
[0033]
Then, signal processing is performed by digital signal processors (DSPs) 27a and 27b, which are transferred and stored in the memory circuit for each channel provided in the memory board 32 on the expansion slot in the computer 31 of the scanning cell measuring apparatus. The
[0034]
A GPIB board 33 that performs GPIB (General Purpose Interface Bus) control based on the IEEE-488 standard is provided in the expansion slot of the computer 31 of the scanning cell measurement device, and a GPIB interface control circuit (GPIB I / O) on the device side is provided. F) Communication is performed between the CPU 31 (central processing unit) 37 on the apparatus side and the computer 31 via the 34.
[0035]
The CPU bus (CPU Bus) on the apparatus side has waveforms for driving the motor controllers 38a and 38b for controlling the drive of the two stepping motors 40a and 40b for driving the X axis and the Y axis of the scanning stage, respectively, and the galvanometer mirror 45. Waveform generation circuit 41 to be generated, digital / analog converters (D / A) 30a and 30b for generating the applied voltage to the cathodes of the respective photomultiplier tubes (PMT) 21a and 21b and controlling the multiplication factor, and the respective photoelectrons Digital / analog converters (D / A) 29a and 29b for generating offset adjustment voltages of photoelectric signals detected by multipliers (PMT) 21a and 21b are connected.
[0036]
Motor drivers 39a and 39b for driving the stepping motors 40a and 40b are connected to the motor controllers 38a and 38b, respectively. The waveform generation circuit 41 includes a digital / analog converter (D / A) 42, a digital / analog converter (D / A) 43 that generates an offset adjustment voltage, and a mixer 44.
[0037]
The computer 31 is an IBM PC / AT or a compatible machine having at least two 16-bit ISA (Industry Standard Architecture) expansion slots below a video graffiti adapter (VGA) board for computer monitor display. Yes.
[0038]
Two slots mount a single memory card 32 and a GPIB board 33. In the scanning cell measuring apparatus according to the present embodiment, the computer is a 4DX-33V type (using Intel CPU, 80486DX-33) PC computer from Gateway (USA), and the GPIB board is National Instruments (USA). A manufactured AT-GPIB type board is used.
[0039]
With the above optical system and electrical system configuration, the operation of the apparatus, such as scanning and signal processing, is comprehensively controlled by the GPIB control command from the computer 31 of the scanning cell measuring apparatus, and the specimen is two-dimensionally scanned with a laser. Thus, a plurality of fluorescence can be detected to obtain scanned image data.
[0040]
Next, the operation of the scanning cell measurement device will be described.
First, the schematic operation of the scanning cell measuring apparatus will be described with reference to the flowchart of FIG.
[0041]
First, the specimen is set on the stage of the scanning cell measuring apparatus (step 1). Next, measurement conditions such as a specimen scanning region, gain, and offset are input to the computer 31 from input means such as a keyboard of the computer 31 (step 2). Then, the computer 31 initializes and creates an image data list (step 3).
[0042]
Next, the scanning area on the set slide glass specimen is divided into small scanning areas limited by the memory capacity of one scanning image memory, and the photoelectric signal is digitized while performing two-dimensional scanning, and the computer The scanned image data is transferred to the memory board 32 on 31 (step 4).
[0043]
Scanned image data for each small scanning region is read by the computer each time, and after the scanned image data is processed by the computer (step 5), the region corresponding to each cell is extracted, and cell information is calculated. Then, a cell data list is created and added (step 6).
[0044]
Then, the measurement ends when the cell data list for the entire predetermined scanning area is created (step 7, step 8). The cell data list obtained as a result of the measurement is saved as a data file, displayed on the computer screen while being subjected to statistical calculation processing such as histogram display, and the examination is completed (step 9, step 10).
[0045]
Next, an image processing process for extracting each cell region / cell information applied to the scanned image data will be described with reference to the flowchart of FIG.
Here, it is assumed that a micronucleus specimen made of blood from an experimental animal is used as the specimen. That is, the entire image of red blood cells is obtained in the scanned image of the second channel that detects amber fluorescence, and the image of micronuclei that appears in the red blood cells is obtained in the scanned image of the first channel that detects green-yellow fluorescence ( step 11).
[0046]
Next, a region exceeding a preset threshold value is extracted in the second channel and recognized as “red blood cells” (step 12, step 13). Here, the image area recognized as “red blood cells” is defined as area A.
[0047]
Next, within the range recognized as “red blood cells”, an area exceeding a preset threshold value in the first channel is extracted and recognized as a micronucleus (step 14). Here, an image region recognized as a micronucleus is referred to as region B.
[0048]
Next, the process in step 15 will be described.
In step 15, for example, if morphological information such as the size of the micronuclei is necessary, the number of scanned image data (number of pixels) in the area exceeding the threshold in the first channel is used as measurement data.
[0049]
When the ratio of the size of micronuclei in erythrocytes is required, it is obtained by dividing (dividing) the number of pixels in the region extracted in the first channel by the number of pixels in the region extracted in the second channel.
[0050]
If quantitative photometric information such as the amount of DNA in the micronuclei is necessary, the sum of the scanned image data of the area exceeding the threshold in the first channel is taken. At this time, for the correction of the background value and the like, the method described in JP-A-3-255365 can be applied, and by adding the correction of the background value and the like, quantitative measurement can be performed more accurately.
[0051]
On the other hand, if only the presence or absence of micronuclei is known, it is only necessary to detect whether or not the threshold is exceeded in the first channel within the range recognized as “red blood cells”. The number of micronuclei in the red blood cell is represented by the number of regions where the data of the first channel exceeds the threshold value.
[0052]
Next, the data of the area A and the area B are recorded for each area A for the calculation result calculated in step 15, the cell data list is added to the data file (step 16), and the image processing is ended (step 17). ).
[0053]
Here, if the purpose of micronucleus specimen measurement is not to obtain quantitative information such as the amount of micronucleus DNA or morphological information such as the size of micronuclei, but to confirm the presence or absence of micronuclei, that is, the appearance rate. For example, it is not necessary to perform quantitative measurement using a scanned image.
[0054]
Therefore, in the scanned image of the first channel for detecting green-yellow fluorescence, it is necessary that the image of the micronuclei appearing in the red blood cells is obtained with good contrast, and as the fluorescence photometric value of each pixel data in the scanned image Quantification is not necessary.
[0055]
Hereinafter, a method for detecting the presence or number of micronuclei in such a case will be described with reference to the flowchart of FIG.
First, a scanned image of the whole red blood cell is obtained in the second channel for detecting amber fluorescence, and an image of micronuclei appearing in the red blood cell is obtained in the first channel for detecting green-yellow fluorescence (step 21).
[0056]
Next, a spatial filter for emphasizing the spot is applied to the scanned image of the first channel (green-yellow fluorescence), and minute micronuclei are enhanced by preprocessing (step 22).
Here, the spatial filter processing for emphasizing the spot is based on a convolution integral of a two-dimensional operator. The size of the operator is set so as to match a spot that is equal to the spot size equivalent to the beam size scanned on the sample, and to match a larger spot with an integer ratio or a power ratio.
[0057]
This makes it possible to optimize spatial filtering for small nuclei that are about the same size as the spot size and detect them sharply, while using a larger operator for slightly larger nuclei. Can optimize spatial filtering and detect sensitively.
[0058]
Next, an image region (referred to as region A) that exceeds a predetermined threshold in the second channel image is extracted (step 23, step 24), and an image that exceeds the predetermined threshold in the first channel image region corresponding to region A A region (referred to as region B) is extracted (step 25).
[0059]
Next, for each area A, the number of areas B in area A is recorded, added to the data file as a cell list, and the image processing ends (step 26, step 27).
That is, a spatial filter process for emphasizing a spot is performed as a pre-process on the scanned image of the first channel, and after the micro-nuclei are emphasized by the pre-process, a region exceeding the threshold is recognized and extracted as a micro-nuclei. The presence or absence of micronuclei, that is, the appearance rate and / or the number of micronuclei that have appeared are output as cell data for each individual red blood cell. Thereby, the presence / absence of minute micronuclei and the detection rate of the number can be increased.
[0060]
In addition, if it is necessary to increase the detection rate of the presence or number of micronuclei and at the same time to measure the DNA amount and size of a somewhat larger micronucleus, while performing the process shown in FIG. A spatial filter process for emphasizing the spot is performed in parallel with the scanned image, and both processing results are output.
[0061]
Hereinafter, with reference to the flowchart of FIG. 6, processing when spot enhancement spatial filter processing is used in combination will be described.
First, a scanned image of the whole red blood cell is obtained in the second channel for detecting amber fluorescence, and a scanned image of micronuclei appearing in the red blood cell is obtained in the first channel for detecting green-yellow fluorescence (step 31).
[0062]
Next, image processing using spot enhancement spatial filter processing is started.
That is, as described in the flowchart shown in FIG. 4 described above, first, an area exceeding the preset threshold value in the second channel (referred to as area A) is extracted and recognized as “red blood cells” (step 32, step 33). .
[0063]
Next, within the range recognized as “red blood cells”, an area exceeding a preset threshold value in the first channel is extracted and recognized as a micronucleus (step 34). That is, an image area (referred to as area B) exceeding the threshold in the first channel image area corresponding to area A is extracted.
[0064]
Next, as described in the flowchart of FIG. 4, the number of pixels (= area) in the region A and the region B, the photometric value data sum (= integrated value), the area ratio between the region A and the region B, etc. are calculated (step 35). ).
[0065]
Next, the calculation results calculated in step 35 are recorded for each region A, the data of region A and region B, the cell data list is added to the data file (step 36), and the image processing is ended (step 37). ).
[0066]
On the other hand, the following processing is performed in parallel with the processing of step 33 to step 36.
First, a spatial filter for emphasizing a spot is applied to the scanned image of the first channel (green-yellow fluorescence), and minute micronuclei are enhanced by preprocessing (step 41). Here, the spatial filter processing for emphasizing the spot is as described in the description of FIG.
[0067]
Next, an image area (referred to as area C) exceeding a predetermined threshold is extracted in the first channel image area corresponding to the image area A extracted in step 33 (step 42). Next, for each area A, the number of areas C in area A is recorded, added to the data file as a cell list, and image processing ends (step 43, step 37).
[0068]
Both processing results are displayed on the display of the computer 31 or the like.
The scanning microscope measuring apparatus according to the present embodiment is configured so that the processing shown in FIGS. 4 to 6 can be arbitrarily switched and can be used depending on the application.
[0069]
Therefore, according to the scanning microscope measurement apparatus of the present embodiment, in the application for confirming the presence / absence rate of small nuclei, the spot emphasis spatial filter is applied to detect small nuclei without omission. Can do.
[0070]
In addition, in applications that measure quantitative information such as micronucleus DNA amount and morphological information such as micronucleus size, extraction of quantitative information and morphological information is performed after threshold processing without applying a spot enhancement spatial filter. By performing the above, more accurate measurement data can be obtained.
[0071]
That is, it is possible to provide a cell measuring apparatus suitable for use in detecting the presence / absence rate of micronuclei in a micronucleus test, and for measuring quantitative information such as the amount of DNA in the micronucleus and the size of the micronuclei.
[0072]
In the description of the above-described embodiment, it was performed based on a micronucleus test specimen in erythrocytes. However, the gist of the present invention is that other uses that combine the quantification / morphometry of the minute fluorescent label appearing inside an ecological structure such as a cell, cell nucleus, or chromosome and the count of appearance rate / number, such as chromosome labeling. Needless to say, the present invention can be similarly applied to the labeling of specific molecular sequences of DNA and DNA, the detection and quantitative measurement of antigens and antibody substances locally found in cells.
[0073]
In addition, even when a red blood cell sample in a micronucleus test is simply stained with PI (Propidium Jodide), detection of cells is performed by detection of forward scattered light instead of fluorescence, and detection of PI is performed only by detection of micronuclei. The micronucleus can be inspected using the method described in this embodiment.
[0074]
【The invention's effect】
As detailed above, according to the present invention, micronuclei of cellsSuch as fine structure or components constituting the structureIt is possible to provide a scanning cell measuring apparatus capable of automatically observing.
[Brief description of the drawings]
FIG. 1 is a diagram showing a multi-parameter fluorescence photometric optical system of a scanning cell measuring apparatus according to an embodiment of the present invention.
FIG. 2 is a diagram showing a configuration of an electric system of the scanning cell measuring apparatus in the same embodiment.
FIG. 3 is a flowchart for explaining a schematic configuration of an operation of the scanning cell measuring apparatus according to the embodiment.
FIG. 4 is a flowchart for explaining an image processing process for extracting each cell region and the like applied to the scanned image data of the scanning cell measuring apparatus according to the embodiment;
FIG. 5 is a flowchart for explaining a method of detecting the presence / absence of micronuclei and the number in the scanning cell measuring apparatus according to the embodiment.
FIG. 6 is a flowchart for explaining processing for performing spot enhancement spatial filter processing in parallel in the scanning cell measurement apparatus according to the embodiment;
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 11 ... Laser, 12 ... Main dichroic mirror, 13 ... Galvanometer mirror, 14 ... Pupil projection lens, 15 ... Objective lens image, 16 ... Objective lens, 17 ... Sample surface, 18 ... Sub dichroic mirror, 19 ... First photoelectron Multiplier tube, 20 ... second photomultiplier tube, 21a, 21b ... photomultiplier tube (PMT), 22a, 22b ... head amplifier, 23a, 23b ... mixer, 24a, 24b ... analog integrator, 25a, 25b ... Analog digital converter, 27a, 27b ... digital signal processor, 31 ... computer, 32 ... memory board, 33 ... GPIB board, 34 ... GPIB interface control circuit, 37 ... CPU, 40a, 40b ... stepping motor, 45 ... galvanometer mirror.

Claims (3)

A light beam scanning means for two-dimensionally scanning a light beam spot on a cell population stained and labeled with a single or a plurality of fluorescent dyes having different spectral characteristics;
First fluorescence detection means for detecting fluorescence that labels a minute structure in the cell or a component constituting the structure, out of fluorescence from the cell excited by the light beam scanned by the light beam scanning means;
Second fluorescence detection means for detecting fluorescence that labels the whole cell among fluorescence from cells excited by the light beam scanned by the light beam scanning means;
Spatial filtering means for performing spatial filtering to emphasize the spot of the light beam with respect to the scanned image obtained by the fluorescence detected by the first fluorescence detection means;
First image area calculation means for calculating a first image area that exceeds a first threshold among the scanned image areas obtained by the fluorescence detected by the second fluorescence detection means;
The scanned image obtained by the fluorescence detected by the first fluorescence detecting means within the first image area calculated by the first image area calculating means is filtered by the spatial filtering means. A second image area that exceeds a second threshold is calculated out of the scanned image areas, and a second image area that exceeds the second threshold is calculated for the scanned image that is not filtered by the spatial filtering means . A second image area based on the scanned image on which the spatial filtering has been performed, and a fine structure in the cell that is less than or equal to the light beam or a structure thereof is configured. The second image region is detected using a second image region based on a scanned image that is detected for the presence or absence of the component and is not subjected to the spatial filtering. Scanning cells measuring apparatus and carrying out a quantitative measurement of the image area. A light beam scanning means for two-dimensionally scanning a light beam spot on a cell population stained and labeled with a single or a plurality of fluorescent dyes having different spectral characteristics;
First fluorescence detection means for detecting fluorescence from a minute fluorescence-labeled portion inside a cell nucleus or a chromosome among fluorescence excited by a light beam scanned by the light beam scanning means;
Second fluorescence detection means for detecting fluorescence that labels the whole of the cell nucleus or chromosome among the fluorescence from the cells excited by the light beam scanned by the light beam scanning means;
Spatial filtering means for performing spatial filtering to emphasize the spot of the light beam with respect to the scanned image obtained by the fluorescence detected by the first fluorescence detection means;
First image area calculation means for calculating a first image area that exceeds a first threshold among the scanned image areas obtained by the fluorescence detected by the second fluorescence detection means;
The scanned image obtained by the fluorescence detected by the first fluorescence detecting means within the first image area calculated by the first image area calculating means is filtered by the spatial filtering means. A second image area that exceeds a second threshold is calculated out of the scanned image areas, and a second image area that exceeds the second threshold is calculated for the scanned image that is not filtered by the spatial filtering means . A second image area based on the scanned image on which the spatial filtering has been performed, and a fine structure in the cell that is less than or equal to the light beam or a structure thereof is configured. The second image region is detected using a second image region based on a scanned image that is detected for the presence or absence of the component and is not subjected to the spatial filtering. Scanning cells measuring apparatus and carrying out a quantitative measurement of the image area. The size of the spatial filtering operator is a size that matches the same spot size as the beam spot scanned on the specimen, and has a lower limit.
The apparatus further comprises storage means for storing the number of second image areas calculated by the second image area calculation means in the first image area calculated by the first image area calculation means. Item 3. A scanning cell measuring apparatus according to item 1 or 2.

Images