WO2003067230A1

WO2003067230A1 - Fluorescent image measuring method and device

Info

Publication number: WO2003067230A1
Application number: PCT/JP2003/001224
Authority: WO
Inventors: Naohiro Noda; Mutsuhisa Hiraoka; Kazuhito Takahashi; Akihito NARIKUNI
Original assignee: Fuji Electric Holdings Co.,Ltd.
Priority date: 2002-02-07
Filing date: 2003-02-06
Publication date: 2003-08-14
Also published as: JP4106626B2; JPWO2003067230A1; AU2003207248A1

Abstract

A fluorescent image measuring method and device in which auto-focusing (AF) is carried out according to image information obtained through imaging means. A sample used as a measurement specimen is irradiated with light for auto-focusing in a fluorescent image measurement wavelength band, and the focused state is judged from the image information obtained by the irradiation. According to the focused state, at least one of a light-receiving optical system and the sample is driven, the focus position is searched for, the irradiation of the auto-focusing light is stopped after the focus position is reached, the sample is irradiated with excitation light, and a fluorescent image formed by the light emitted from the specimen is measured. Thus AF can be carried out according to image information by means of a simple structure having a small number of constituent elements without causing extinction for even a sample of low fluorescence intensity.

Description

Description ^; Fluorescence image measurement method and device

This invention fluorescently labels (stains with a fluorescent reagent) cells such as microorganisms and tissue cells and fine particles such as mineral particles, and generates fluorescence by exciting the reagent. The present invention relates to a fluorescence image measurement method and apparatus for generating fluorescence by exciting fluorescent molecules originally present in fine particles such as mineral particles and measuring the fluorescent image. Background art

Fluorescence image measurement of microorganisms and tissue cells is used in many fields dealing with living organisms. In such image measurement, autofocus (AF) technology has come into general use with the development of image sensors and image processing technology in recent years. In general auto focus, the amount of high frequency components (contrast) contained in an image signal is determined, and the position where the contrast is maximized is determined as the focal point.

In the present invention, the microorganisms include prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast, fungi, lower algae, viruses, and the like.The tissue cells include animal and plant-derived cultured cells and It contains pollen such as cedar and cypress. In addition, it is not limited only to living things, such as measuring dead bacteria. Further, in the measurement method of the present invention, staining can be performed with one or more color-forming substances capable of staining a detection target (for example, a microorganism). The color-forming substance is not particularly limited as long as it is capable of forming a color by acting on cell components contained in a microorganism, and typical examples thereof include nucleic acids and proteins. And a fluorescent staining solution for staining More specifically, in the case of microbes in general, when analyzing microorganisms in general, it can be used to analyze the structure of fluorescent nucleobase analogs, fluorescent dyes that stain nucleic acids, blue liquids that stain proteins, and proteins. Environmentally friendly fluorescent probes used, stains used for analysis of cell membrane and membrane potential, stains used for fluorescent antibody labeling, etc.For aerobic bacteria, stains that develop color by cell respiration are used. In the case of eukaryotic microbes, stains that stain mitochondria, stains that stain the Golgi apparatus, stains that stain the endoplasmic reticulum, a stain that reacts with intracellular esterase and its modifying compounds, etc. When targeting higher animal cells, a staining solution used for observing bone tissue, a staining solution that is a nerve cell tracer, and the like can be mentioned.

After staining the microorganisms or the like with the staining reagent, the reagent is excited to generate fluorescence, and the fluorescence image is measured by an imaging device (for example, a CCD camera device), whereby the microorganisms and the like can be counted. The color of the generated fluorescence (green, red, etc.) or the wavelength band of the fluorescence differs depending on the microorganism and the type of the staining reagent.

By selecting the type of the chromogenic substance, a total cell count detection for detecting all microorganisms, an assay for staining and counting only microorganisms having respiratory activity, an assay for staining and counting only microorganisms having esterase activity, Alternatively, it can be applied to a wide range of fields, such as an assay that stains and counts microorganisms of a specific genus or species by using a multiple staining method that combines multiple chromogenic substances.

By the way, in the measurement of microorganisms and tissue cells, a method of labeling a sample with a fluorescent reagent is widely used, but in this case, there are the following problems. First, since the amount of fluorescence in the measurement target is often small, when performing AF on a fluorescence image, the incident light amount that can be detected by the AF image sensor is obtained. This requires a long accumulation time, and as a result, the time required for AF becomes longer. In addition, the sample is quenched by the excitation light irradiation during AF, and the brightness decreases. In extreme cases, measurement becomes impossible.

A focus detection device for a microscope has been proposed which solves the above problems and aims at improving the efficiency of autofocusing (for example, see Patent Document 1 described later).

According to the description of the publication, the focus detection device for a microscope disclosed in Patent Document 1 states that “the incident light fluorescence that irradiates a sample with excitation light of a specific wavelength and collects light longer than the excitation light with an objective lens. A filter that transmits light having a wavelength longer than the fluorescent light in the observation means and the transmitted illumination means, a dichroic mirror that transmits one of the lights transmitted through the filter and reflects the other, and a dich-opening mirror. The image sensor is arranged on the short wavelength side and the long wavelength side, respectively, which are separated by the image sensor and captures the observation light image of the sample, and an image sensor for accumulating the observation light image of the sample. Focus detection means for detecting the focus state of the observation light image, servo means for driving at least one of the objective lens and the sample side according to the degree of focus of the focus detection means, and a focus means and an image Capacitors, Oh in those with C P U to control the focus detection means "0.

That is, the apparatus disclosed in Patent Document 1 described above is provided with a transmissive illumination means on the side facing the objective lens, and irradiates the specimen with light having a wavelength longer than the fluorescence observation wavelength by this transmissive illumination, thereby obtaining the specimen. It performs AF based on the observed image, has wavelength separation means for separating the fluorescence image and the AF image, and has two systems of imaging means for obtaining each image. In addition, when fluorescence image measurement is performed in a plurality of wavelength bands by switching the fluorescence image measurement wavelength, an in-focus position is adjusted by combining an optical path length correction unit. However, the one disclosed in Patent Document 1 has various problems as described below. That is,

1) Not only are there many optical filters and mirrors, but also multiple image sensors and optical path length correction units are required, which complicates the configuration and increases costs.

2) Since there are many optical elements and the light receiving efficiency is low, it is difficult to measure samples with low brightness.

3) Since the image measured by the transmitted illumination is used for AF, it is impossible to focus on a sample captured on the surface such as a membrane filter (filter for sample, filter for capturing) that does not transmit light.

4) AF is difficult for specimens with unclear contrast and unclear focus targets.

Further, unlike Patent Document 1, the method does not detect the focus state of an observation light image of a specimen, but Patent Document 2 described below discloses an optical measurement method for accurately focusing an optical system. Describes the following method.

According to the optical measurement method disclosed in Patent Document 2, according to the description of the publication, “a plurality of excitation light sources having different wavelength regions are provided, and a light is generated from a first excitation light source among the plurality of excitation light sources. In the optical measurement method of irradiating the sample with the first excitation light thus obtained and detecting the fluorescence generated from the sample by the irradiation through a lens, a wavelength region substantially equal to the wavelength of the fluorescence is selected from the plurality of excitation light sources. Selecting a second excitation light source provided, irradiating the sample with second excitation light generated from the second excitation light source, detecting the second excitation light reflected by the sample through the lens, An optical measurement method, wherein focusing is performed by adjusting the position of the lens so that the detected value is maximized.

The optical measurement method disclosed in Patent Document 2 is mainly for reading a DNA array test piece, and the position where the amount of reflected light is maximum is defined as a focus position. As described above, autofocus is not performed by detecting the focus state of the observation light image of the specimen or based on image information. In addition, in order to calibrate the amount of reflected light, it is necessary to irradiate the light for autofocus on the same optical path as the excitation light, and for that purpose, it must be incident by a coaxial epi-optical system. Further, as is apparent from FIG. 4 of the publication and the description of the relevant part, there is a problem that the device configuration is complicated and the cost is high, for example, it is necessary to rotate a plurality of types of dichroic mirrors with a motor.

(Patent Document 1)

Japanese Patent Application Laid-Open No. 2001-91182 (page 114, FIG. 1)

(Patent Document 2)

SUMMARY OF THE INVENTION The present invention has been made in view of the above points, and has a simple configuration having a small number of constituent elements. A fluorescence image measurement method that enables AF based on image information without quenching even for specimens with low fluorescence intensity, and for specimens supplemented on the membrane filter surface or specimens with unclear contrast. An object of the present invention is to provide a fluorescence image measurement device. Disclosure of the invention

In order to solve the above-mentioned problems, the invention according to claim 1 provides a fluorescent image measurement method for performing autofocus based on image information obtained via an imaging unit. First, irradiate autofocusing light emitted in the fluorescence image measurement wavelength band, determine the degree of focusing based on the image information obtained as a result, and determine the degree of focusing based on the degree of focusing. Drive the at least one of them to search for the in-focus position, and after arriving at the in-focus position, stop irradiating the auto-focusing light. Then, irradiate the specimen with excitation light, and obtain a fluorescence image emitted from the specimen. Feature to measure And As a result, autofocus can be performed without irradiating the sample with excitation light, and autofocus can be performed using a simple configuration with few components, so that a decrease in light-receiving efficiency is minimized, and Thus, it is possible to measure a specimen having a low fluorescence intensity.

As an embodiment of the invention described in claim 1, the inventions in claims 2 to 8 described below are preferable. That is, the measurement sample is a cell such as a microorganism or a tissue cell (the invention of claim 2).

3. The fluorescence image measurement method according to claim 1, wherein the autofocus light is on the same side as the excitation light irradiation side with respect to the specimen, and further includes an excitation light irradiation axis. Irradiation is performed from the direction of the irradiation axis having a predetermined inclination angle (the invention according to claim 3). This simplifies the device configuration, and enables autofocusing of a sample captured on the surface of the membrane filter.

Furthermore, in the fluorescence image measurement method according to claim 2, the measurement sample includes a plurality of types of cells, and when the plurality of types of cells are measured, the measurement sample is set in advance according to the cells to be measured. The autofocus light in a plurality of fluorescence image measurement wavelength bands is sequentially switched and irradiated to determine the degree of focusing, and measure a fluorescence image of each cell (claim 4). According to this, the present invention can be applied to a system that performs image measurement with a plurality of excitation-fluorescence characteristics by switching light for autofocus.

In any one of the first to fourth aspects of the present invention, a pattern close to the specimen may be provided, and autofocus may be performed based on image information of the pattern. Invention of range 5 :). For AF, the contrast is evaluated.However, malfunction such as focusing on a position other than the sample holding position is prevented, and the contrast evaluation is performed. P Hiring 224

The method using patterns is effective for accurate and convenient flight.

In the invention according to claim 5, the pattern can be provided on a surface of a slide glass holding a specimen (the invention according to claim 6). In the invention according to claim 5, the pattern can be provided on a surface of a filter that filters and supplements the sample (the invention according to claim 7). Further, in the invention of claims 5 to 7, the pattern includes a number or a figure, and by identifying these, it is possible to obtain positional information of the specimen. (Invention of paragraph 8). Further, as an apparatus for performing the fluorescence image measurement method, the inventions of the following claims 9 to 11 are preferable. That is, in a fluorescence image measurement device that performs autofocus based on image information obtained through an imaging unit, an autofocus light irradiation unit that irradiates a sample with light in a fluorescence image measurement wavelength band; Excitation light irradiating means, autofocus and fluorescence image measurement imaging means, fluorescence and excitation filters and a fluorescent filter block having a dichroic mirror, and a light receiving optical system or sample according to the degree of focus Focusing driving means for driving at least one of the following; and arithmetic control means, wherein the arithmetic control means determines the degree of focus based on image information obtained by irradiating the autofocus light, At least one of the receiving optical system or the sample is driven according to the degree of focus to search for the in-focus position. Stopping the irradiation, then, irradiated with excitation light to the specimen, characterized in that it comprises a control function of measuring the fluorescence image emitted by the sample (the invention of Claim C. Section 9).

In the ninth aspect of the present invention, the light source in the autofocus light irradiating means and / or the excitation light irradiating means is a light emitting diode or a semiconductor laser. . Further, in the invention according to claim 9, in order to perform autofocus by switching light in a plurality of fluorescence image measurement wavelength bands, the autofocus light irradiation unit includes a plurality of autofocus light irradiation units. A light source, and a plurality of the fluorescent filter blocks are provided; and the arithmetic control means includes a light source of a plurality of predetermined fluorescence image measurement wavelength bands respectively corresponding to a plurality of types of cells. And a function of performing auto focus by sequentially switching (invention of claim 11). BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic configuration diagram of a fluorescence image measurement device according to a first embodiment of the present invention.

FIG. 2 is a schematic configuration diagram of a fluorescence image measurement device according to a second embodiment of the present invention.

FIG. 3 is an explanatory diagram illustrating an example in which a pattern for AF is provided.

FIG. 4 is an explanatory diagram illustrating a different example in which an AF pattern is provided. FIG. 5 is an explanatory diagram illustrating still another example in which positional information is provided in an AF pattern. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic configuration diagram of a fluorescence image measurement device according to a first embodiment of the present invention.

In Fig. 1, 1 is a sample, 2 is a light source for AF, 3 is a dichroic mirror, 4 is a filter, 5 is an objective lens, 6 is an imaging lens, 7 is an image sensor, 8 is a calculation unit, and 9 is a stage movement. 10 is a light source for excitation, 11 is a condenser lens, and 13 is a fluorescence filter block.

In the measuring device shown in Fig. 1, the sample 1 is irradiated obliquely from above. In this example, a method of performing AF with incident light and performing fluorescence image measurement using an epi-illumination optical system is not limited to this method.For example, a modification in which excitation light and AF light are irradiated from an oblique direction, Includes an example in which the light for AF is transmitted light, but the structure shown in FIG. 1 is the simplest in structure and can reduce the cost. As in the conventional AF, a general method using contrast between pixels is used.

In FIG. 1, first, the specimen 1 is irradiated from the AF light source 2 with light containing a wavelength in the fluorescence image measurement wavelength band, so as to suppress the fading of the specimen. The fluorescence image measurement wavelength band is determined by the spectral transmission characteristics of the dichroic mirror 3 and the spectral transmission characteristics of the fluorescent light receiving filter 4.

As the AF light source 2, a light emitting diode (LED) or a semiconductor laser is suitable. The reason is that the light emission spectrum can be variously selected by selecting the element, and the characteristics are not easily deteriorated even if ONZOFF is repeated. Furthermore, because these elements are small and lightweight, they can be easily assembled as light sources for AF.

Since an image for AF can be obtained in the fluorescence image measurement wavelength band, a wavelength separating unit and two image pickup devices as in the device disclosed in Patent Document 1 are not required, and the devices described in Patent Documents 1 and 2 are required. In comparison, the device configuration can be simplified. It is also possible to obtain a desired wavelength by using a white lamp as the light source for AF and combining an optical filter, and to perform irradiation and non-irradiation by opening and closing the shutter. Further, the angle of irradiation of the AF light is preferably such that the inclination angle with respect to the sample surface is about 5 to 45 °.

The image of the specimen at the time of irradiating the AF light is captured by the image sensor 7 via the objective lens 5, the dichroic mirror 13, the fluorescent light receiving side filter 4, and the imaging lens 6. As the imaging element, a CCD camera element or a CMOS camera element is suitable. The image obtained by the image sensor 7 is used as the arithmetic control means. Is sent to the calculation unit 8, where the contrast is evaluated. The contrast is evaluated by a general AF method, for example, calculating as a luminance difference between adjacent pixels, and setting a position where the contrast becomes maximum as a focal point position. The processing up to the in-focus position is generally as follows.

1) The specimen is moved by the stage moving mechanism 9

2) Image capture

3) Contrast evaluation

4) Compare previous screen with contrast

5) If contrast is increasing, move further in the same direction

6) If contrast is decreasing, move in the opposite direction

When actually performing AF, in addition to the above, in addition to the above, first scan the entire area roughly to grasp the approximate focus position, gradually reduce the moving distance as it approaches the focus position, or set the same position It is necessary to have an algorithm such as judging the focus position after reciprocating a number of times. It should be noted that the stage moving mechanism 9 is not an essential requirement, as long as it can search the focal point by driving at least one of the sample 1 and the light receiving system.

After reaching the focal point, the AF light is turned off, and then the excitation light source 10 is turned on. The light from the excitation light source 10 irradiates the sample 1 via the condenser lens 11, the excitation side filter 12, the dichroic mirror 3, and the objective lens 5, thereby enabling fluorescence image measurement. .

Conventionally, a high-pressure mercury lamp is often used as the excitation light source. However, the characteristics of the high-pressure mercury lamp deteriorate significantly when ONZNZ OFF is repeated in a short time. A shutter is required. Therefore, if the wavelength characteristics and the amount of light can be satisfied, it is desirable to use a light emitting diode (LED) or a semiconductor laser as the excitation light source, similarly to the AF light source. 0301224

11

One example of suitable specific data (examples of excited blue light-fluorescent observation green light-AF green light) of various main members in the apparatus shown in Fig. 1 is shown below.

1) Light source for excitation: Blue LED with center wavelength of 470nm

2) Transmission wavelength band of excitation light side optical filter: 450 ~ 470nm

3) Characteristics of the dichroic mirror: the wavelength of the branch point between the reflectance and the transmittance is 505 nm (that is, the transmittance is about 50% at 505 nm, and the transmittance decreases at wavelengths shorter than 505 nm (reflectance) Has a mirror characteristic of increasing the transmittance (reducing the reflectance) at wavelengths longer than 505 nm)

4) Transmission wavelength band of fluorescence observation side optical filter: 510 ~ 560nm

5) Light source for AF: Green LED with center wavelength 535nm

Next, a fluorescence image measurement device according to a second embodiment of the present invention shown in FIG. 2 will be described. 2, the same functional members as those shown in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted.

FIG. 2 shows a case where a plurality of living cells, for example, a plurality of bacteria are simultaneously measured, or a case where the same bacterium contains live and dead bacteria. 1 shows an apparatus according to the invention of item 1. For example, when simultaneously measuring a plurality of bacteria, a plurality of staining reagents are used depending on the type of bacteria, and the fluorescent color differs depending on the type of bacteria and the staining agent.

Therefore, in the case of the fluorescence image measurement system for measuring a plurality of living cells as described above, the three elements of the excitation side filter 12, the dichroic mirror 3, and the image measurement side filter 4 are usually combined into one unit. As shown in FIG. 2, a plurality of fluorescent filter blocks 13 are provided. By switching these, it is possible to perform image measurement with multiple excitation-fluorescence characteristics using white light emitted from a high-pressure mercury lamp. An example in which the present invention is applied to a fluorescence image measurement system having such an image measurement condition switching mechanism will be described below with reference to FIG. In FIG. 2, for example, a position recognition mechanism 14 is provided for five fluorescent filter blocks 13, and when a block is switched, its output is taken in, so that the calculation unit 8 automatically recognizes the block setting status. To do it. Further, the calculating unit 8 determines the set fluorescence image measurement wavelength of the fluorescence filter block 13 and irradiates the sample 1 with AF light suitable for the wavelength. As the AF light source 15, a plurality of types of light-emitting diode semiconductor lasers may be switched and used, or a switching mechanism of a white light source and an optical filter having a predetermined spectral transmittance and a shutter mechanism may be combined. May be used.

After reaching the focal point, turn off the AF light. Subsequently, the excitation light source 10 is turned on, or the excitation light source is turned on in advance, and the specimen 1 is irradiated with the excitation light by opening the shirt that has blocked the excitation light, and fluorescence image measurement is performed. The excitation light source may be, in addition to the white light, a plurality of LEDs that emit excitation light according to the measurement sample.

Next, an example in which an AF pattern is provided based on FIGS. 3 to 5 will be described. In the fluorescence image measurement device shown in FIGS. 1 and 2, the contrast is evaluated for A F. At this time, as described above, it is effective to use a pattern to prevent a malfunction such as focusing on a position other than the main holding position and to easily and accurately perform contrast evaluation. First, an example of applying a pattern to the surface of a slide glass holding a specimen will be described with reference to FIG.

As shown in the figure, a pattern is drawn on the surface of the slide glass G in advance. The pattern is drawn using non-fluorescent paint or non-fluorescent metal deposition. The thickness and density of the pattern shall be set so that at least a part of the boundary appears in the field of view when the image is captured.

Specimens such as fluorescently labeled microorganisms and tissue cells are dropped on slide glass, covered with a cover glass, and used as a measurable sample. At this time, the specimen 1224

13

Is held down by the cover glass, and comes into close or close contact with the pattern on the slide glass surface. Set the sample in the fluorescence image measurement system,

Irradiate light for AF. Since at least a part of the pattern is shown in the AF image, autofocus is performed with that part as the target. Subsequently, the excitation light source is turned on and fluorescence image measurement is performed in the same manner as in the embodiment shown in FIGS.

In order to perform the above measurement, it is assumed that the pattern, which is the reference of the autofocus, and the sample are close to or close to each other. In practice, the distance between the pattern and the specimen should be shorter than the depth of focus of the light receiving system composed of the objective lens, lens barrel, imaging lens, and image sensor.

Next, FIG. 4 will be described. When measuring microorganisms and tissue cells, the process of capturing a sample by filtration through a membrane filter and measuring it is an extremely common operation. Hereinafter, a fluorescent image measurement method for providing a pattern on the surface of the membrane filter will be described with reference to FIG.

Similar to the case of Fig. 3, the pattern is drawn on the surface of the membrane filter F by depositing a non-fluorescent paint or a non-fluorescent metal. The thickness and density of the pattern should be set so that at least some of the boundaries are visible in the field of view when the image is captured.

Samples of fluorescently labeled microorganisms and tissue cells are filtered through a membrane filter. The sample captured on the membrane filter is used as a sample for measurement. At this time, the sample is in close proximity to the surface of the membrane filter.

Set the sample in the fluorescence image measurement system and irradiate it with AF light. Since at least a part of the pattern is reflected in the AF image, autofocus is performed with that part as the target. After reaching the in-focus position, turn off the AF light. Subsequently, the excitation light source is turned on and fluorescence image measurement is performed. Now.

In a device that performs autofocus based on a transmitted light image as in the device disclosed in the above-mentioned Patent Document 1, in particular, for a sample having no coloration and a refractive index close to water, the differential interference method is used. Image measurement using is an essential requirement. As described above, when a microorganism is measured, the operation of supplementing the sample by filter filtration and measuring the sample is an extremely well performed operation, but in this state, it is difficult to obtain a differential interference image. That is, what is disclosed in the gazette of Patent Document 1 cannot be applied to the sample supplemented on the filter.

Next, FIG. 5 will be described. When measuring specimens such as microorganisms and tissue cells, it is not uncommon to scan a sample on a plane perpendicular to the fluorescence image measurement direction and measure the image on multiple screens in order to reduce statistical variations. In such a case, if the pattern is not a simple line, but a form that can obtain positional information, for example, as shown in Fig. 5, for example, by drawing a combination of sections and serial numbers on the surface of the membrane filter F, The location of a specimen such as an object or a tissue cell can be grasped.

If microorganisms or tissue cells change over time, this method can be used to recognize individual specimens individually and track those changes. Typical examples are applications that capture the growth of microorganisms and cases where the effects of drugs on tissue cells are evaluated. Such positional information may be formed on the surface of the slide glass. Industrial applicability

As described above, the present invention can be used for a measurement method and an apparatus for labeling a sample containing fine particles such as cells such as microorganisms and tissue cells and mineral particles with a staining reagent and measuring a fluorescent image emitted from the sample. Main measurement method and apparatus The fields of application include medical care, food production, and water and sewage.

According to the present invention, autofocus can be performed without irradiating the sample with excitation light, and a reduction in light receiving efficiency can be minimized by realizing a simple mechanism with few components. In particular, it can measure samples with low fluorescence intensity.

In addition, since AF light is irradiated from the direction of the excitation light irradiation axis that is on the same side as the excitation light irradiation direction and has a predetermined angle of inclination with respect to the excitation light irradiation axis, auto-focusing is also possible for samples supplemented on the membrane filter surface. is there.

Further, according to the method including a plurality of AF light sources, the present invention can be applied to a system that performs image measurement with a plurality of excitation-fluorescence characteristics while switching the fluorescence filter block. Furthermore, even for a sample whose contrast is unclear, autofocus can be performed by using a pattern provided close to the sample.

Claims

The scope of the claims

1. In a fluorescence image measurement method for performing autofocus based on image information obtained through an imaging unit,

A sample as a measurement sample is first irradiated with autofocus light emitted in a fluorescence image measurement wavelength band, and the degree of focus is determined based on the image information obtained as a result. At least one of the light receiving optical system and the sample is driven to search for the focus position, and after reaching the focus position, irradiation of the autofocus light is stopped.After that, the sample is irradiated with excitation light. A fluorescence image measurement method, comprising: measuring a fluorescence image emitted from a sample.

2. The fluorescence image measurement method according to claim 1, wherein the measurement sample is a cell such as a microorganism or a tissue cell.

3. The auto-focusing light is irradiated onto the specimen from the same side as the excitation light irradiation side and from an irradiation axis direction having a predetermined inclination angle with respect to the excitation light irradiation axis. 3. The fluorescence image measurement method according to claim 1 or 2, wherein

4. The measurement sample includes a plurality of types of cells, and when measuring the plurality of types of cells, the auto-focusing light of a plurality of fluorescence image measurement wavelength bands preset in accordance with the cells to be measured is sequentially applied. 3. The fluorescence image measurement method according to claim 2, wherein the degree of focus is determined by switching and irradiating, and a fluorescence image of each cell is measured.

5. The fluorescent image according to any one of claims 1 to 4, wherein a pattern close to the specimen is provided, and autofocus is performed based on image information of the pattern. Measurement method.

6. The pattern should be provided on the surface of the slide glass holding the specimen 6. The fluorescence image measurement method according to claim 5, wherein:

7. The fluorescence image measurement method according to claim 5, wherein the pattern is provided on a surface of a filter that filters and captures the sample.

8. The pattern according to any one of claims 5 to 7, wherein the pattern includes a number or a figure, and by identifying the pattern, position information of the sample can be obtained. The fluorescent image measurement method according to the above section.

9. In a fluorescence image measurement device that performs autofocus based on image information obtained through an imaging unit,

Autofocus light irradiation means for irradiating the sample with light in the fluorescence image measurement wavelength band, excitation light irradiation means for the sample, imaging means for autofocus and fluorescence image measurement, and fluorescence and excitation A fluorescent filter block having a filter and a dichroic mirror, focusing driving means for driving at least one of the light receiving optical system or the sample according to the degree of focusing, and arithmetic control means. ,

The arithmetic and control means determines the degree of focus based on image information obtained by irradiating the autofocus light, and drives at least one of the light receiving optical system or the sample according to the degree of focus to obtain the focus. A control function for searching for a focus position, stopping irradiation of the autofocus light after reaching the in-focus position, irradiating the sample with excitation light thereafter, and measuring a fluorescence image emitted from the sample. Fluorescent image measurement device.

10. The fluorescence image measurement device according to claim 9, wherein the light source for the autofocus light irradiation means and z or the excitation light irradiation means is a light emitting diode or a semiconductor laser.

1 1. In order to perform autofocus by switching light in a plurality of fluorescence image measurement wavelength bands, the autofocus light irradiation means has a plurality of autofocus light sources, and a plurality of the fluorescent filter blocks. Provided Further, the arithmetic and control unit has a function of sequentially switching light sources of a plurality of predetermined fluorescence image measurement wavelength bands respectively corresponding to a plurality of types of cells to perform autofocus. 10. The fluorescence image measurement device according to claim 9, wherein