JP5010890B2 - Biological sample analysis method - Google Patents

Description

  The present invention relates to a biological sample analysis method for analyzing biological samples such as cells, tissues, and individuals.

  Patent Document 1 discloses a microorganism detection method and the like. Specifically, an apparatus for capturing a target microorganism using a substrate material combined with a substance that specifically captures the target microorganism present in a sample and optically detecting the target microorganism captured on the substrate material And a method are disclosed.

  Non-Patent Document 1 discloses a method of observing the state of infection in a tissue by fluorescently labeling the microorganism and a method of observing the infection of the microorganism using an electron microscope.

JP 2005-172680 A Fengwu Li et al. , “Plasmodium Okinate-secreted Proteins Secreted Through a Common Microphone Way of Targets of Blocking Maria Transmission”, J. Biol. Chem. , Vol. 279, no. 25, Jun 18, pp. 26635-26644, 2004

  However, according to Patent Document 1, since non-tissues such as blood, feces, and food are targeted and specific microorganisms are captured therefrom, the microorganisms in the tissue are allowed to exist in the tissue without being removed from the tissue. There was a problem that it could not be detected in the state.

  Further, according to Non-Patent Document 1, since fluorescence is used, the background due to autofluorescence is high and noise is large, so that there is a problem that microorganisms in the tissue cannot be clearly captured. Further, according to Non-Patent Document 1, since fluorescence is used, laser permeability is low for a thick tissue, so that there is a problem that microorganisms in the thick tissue cannot be clearly captured. there were. Further, according to Non-Patent Document 1, since fluorescence is used, there is a problem that damage to the tissue due to laser irradiation cannot be avoided. Further, according to Non-Patent Document 1, since an electron microscope is used, there is a problem that the apparatus and facilities become large and the tissue cannot be observed in a live state.

The present invention has been made in view of the above problems, and can capture a biological sample such as a thick living tissue clearly without damaging the biological sample. It is an object of the present invention to provide a biological sample analysis method capable of analyzing a sample accurately and in detail.
Another object of the present invention is to provide a biological sample analysis method capable of analyzing a biological sample with a simple apparatus configuration.

To solve the above problems and achieve the object, BIOLOGICAL sample analyzing method according to the present invention, at least one of a host, emitted from at least one at least one of the plurality of biological samples is a microorganism An imaging process for capturing at least one luminescent image of the biological sample by selectively detecting the weak light, and an analysis process for analyzing the biological sample based on the luminescent image captured in the imaging process. It is characterized by including.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, in the imaging step, imaged repeatedly the luminescent image, and in the analysis step, a change in the biological sample based on the plurality of the luminescent image It is characterized by analyzing.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, in the imaging step, in addition to the luminescent image, further captures a fluorescence image of the bright-field image and / or the biological sample of the biological sample In the analyzing step, the biological sample is analyzed based on a superimposed image that is an image obtained by superimposing the light-emitting image captured in the imaging step and the bright field image and / or the fluorescent image. To do.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, in the imaging step, the luminescent image and imaged repeatedly the bright field image and / or the fluorescence image, and in the analysis step, a plurality of the The change of the biological sample is analyzed based on the superimposed image.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, in the imaging step, in a cooled charge-coupled light detecting element, characterized by imaging at least the luminescent image.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, in the imaging step, represented by numerical aperture (NA) and projection magnification (β) (NA ÷ β) 2 value of 0.01 Using the objective lens as described above, at least the emission image is captured.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, the value of the (NA ÷ β) ² of the objective lens is characterized in that at 0.039 or more.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, the value of the (NA ÷ β) ² of the objective lens is characterized in that at 0.071 or more.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, the host cell, and wherein the tissue is any one of an individual.

Further, BIOLOGICAL sample analyzing method according to the present invention, in the above invention, the microorganism is characterized in that it is one having a bioluminescent protein.

  In the present invention, at least one biological sample is obtained by selectively detecting weak light emitted from at least one of a plurality of biological samples in which at least one is a host and at least one is a microorganism (the other is). Since the biological sample is analyzed based on the captured luminescent image, it can be clearly captured without damaging the biological sample, and as a result, the biological sample can be analyzed accurately and in detail. There is an effect that can be done. In addition, the present invention has an effect that a biological sample can be analyzed with a simple apparatus configuration.

  Embodiments of a biological sample analysis method according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  First, in the present invention, at least one type of host and at least one type of microorganism are targeted as biological samples. In the present invention, the subject to be imaged may be at least one of at least two different biological samples. For example, a combination of an animal or plant tissue and one or more types of microorganisms that infect it can be used. By using these objects, it is possible to detect a state in which a microorganism is infected. Further, examples include a combination of animal or plant tissue and one or more kinds of microorganisms having a genetic mutation that infects it. Microorganisms that have undergone genetic mutation are microorganisms in which the expression level of a specific gene is adjusted by commonly used genetic manipulations such as knockout genes, chimeric genes, specific protein overexpression genes, and RNA interference. By using the microorganism, it is possible to detect the effect of the gene whose expression is regulated on the tissue and / or microorganism.

  Further, in the present invention, the faint light refers to an extremely faint light signal that is typically invisible to the naked eye and represents a genetic expression level due to bioluminescence. In addition to the bioluminescence, the weak light in the present invention includes fluorescence, chemiluminescence, and the like that emit a weak light signal at the same level as the bioluminescence. For example, fluorescence in BRET (bioluminescence resonance energy transfer) is emitted by the weak energy of bioluminescence by the substrate, and is therefore included in bioluminescence. In the present invention, since at least two types of biological samples are targeted, if at least one type of biological sample generates faint light, the faint light may be detected and imaged. If an optical signal that can be identified for a sample, that is, faint light is generated, the faint light may also be detected and imaged.

Next, the configuration of a biological sample analyzer 100 which is an apparatus for carrying out the biological sample analysis method according to the present invention will be described with reference to FIG. FIG. 1 is a diagram showing the configuration of the biological sample analyzer 100. As shown in FIG. 1, a biological sample analyzer 100 includes a sample stage 102, a heat plate 104, a plate temperature controller 106, a gas supply mechanism 108, a water supply mechanism 110, an objective lens 112, and an objective lens heater. 114, a heater temperature controller 116, an objective lens Z-axis drive mechanism 118, an infrared cut filter 120, a CCD camera 122, a CCD camera cooling device 124, and a personal computer 128. The sample stage 102, the heat plate 104, the objective lens 112, the objective lens heater 114, the objective lens Z-axis drive mechanism 118, the infrared cut filter 120, the CCD camera 122, and the CCD camera cooling device 124 include a light shielding box 130 with a lid as shown in the figure. Covered with. The light shielding box 130 with a lid is made of a light shielding material such as aluminum, for example, and the surface of the member is subjected to a black coating process so that light outside the light shielding box with the lid does not enter the inside. . An openable / closable lid 130a is attached to the upper surface of the light shielding box 130 with a lid via a hinge. The experimenter opens and closes the lid 130a to install and remove the sample S.
The configuration essential in the present embodiment is represented by optical conditions (specifically, numerical aperture (NA) and projection magnification (β) (NA ÷ β), which will be described later. ) The objective lens 112 that satisfies the condition ² ), an appropriate light shielding environment such as the light shielding box 130 with the lid, and the CCD camera 122 are simple.

  The sample stage 102 is for placing the sample S, and a heat plate 104 is placed thereon. The sample stage 102 can be moved in the horizontal direction (XY direction) by a screw operation. A water tank T is disposed on the heat plate 104. The heat plate 104 is connected to a plate temperature controller 106, and an environmental temperature (for example, about 37 ° C.) suitable for the growth of microorganisms is set at intervals of 0.5 ° C. by the plate temperature controller.

In the water tank T, a sample container C, which is a container for holding the sample S, is disposed, and pure water W is stretched to maintain the humidity in the sample container C. A sample S is arranged in the sample container C. An enlarged view of the periphery of the sample container C is shown in FIG. FIG. 2 is an enlarged view around the sample container C. FIG. The sample S is put in a sample container C made of a petri dish or the like and immersed in the culture solution Cs. A sample container cover Cv is attached to the sample container C to prevent the sample S in the sample container C from drying and dust from being mixed into the sample container C. Returning to FIG. 1, at least the bottom surface of the sample container C is optically transparent so that a normal objective lens can be used, and is the same material as the microscope cover glass and has a thickness of 0.17 mm. The pure water W stretched in the water tank T is supplied through a nozzle 110 a constituting the water supply mechanism 110. Above the water tank T, a mixed gas in the gas cylinder 108a which constitutes the gas supply mechanism 108 (CO ₂ 5%, the O ₂ gas containing 95%), through the gas supply tube 108b that constitute the gas supply mechanism 108 It is supplied at a flow rate of 50 mL / min.

  Here, the sample S is a biological sample including a microorganism having a bioluminescent protein, and is a host for the microorganism. Hosts include individuals, tissues, cells and the like. The individual is an animal or a plant. Thereby, the ecology of microorganisms can be analyzed for various objects. The individual is an animal or a plant. Animals are all those that can be infected by microorganisms, such as mammals and insects. Plants are all those that can be infected by microorganisms, such as tobacco, tomatoes and eggplants. Tissues are anything that can be infected by microorganisms, such as the brain, kidneys, and intestines. The cells are all those that can be infected by microorganisms, and examples thereof include cultured cells such as HeLa cells and CHO cells, and cells collected from individuals or tissues such as primary neurons. Further, the sample S may be surgically removed, or may be a part of an individual that is surgically exposed without being extracted. If the microorganism is selectively imaged, the sample S is not necessarily alive, and only the microorganism needs to be alive in the sample S. In the case where the microorganism is selectively imaged, the microorganism is preferably placed in an appropriate culture environment by the components in the sample S or the culture solution.

  Several types of bioluminescent proteins are known. For example, typical organisms containing bioluminescent proteins of the luciferase system (luc) are fireflies and beetles, which emit green, yellow-green, yellow, orange light, among which orange light This is preferred because it has a wavelength that penetrates tissue more easily than light with a short wavelength. In order to cause the luciferase enzyme to emit light, a luminescent substrate must be supplied to the luciferase enzyme. The luminescent substrate to be used is selected according to the bioluminescent protein. For example, when the bioluminescent protein is firefly luciferase, luciferin is selected as the luminescent substrate, and when the bioluminescent protein is Renilla luciferase, coelenterazine is selected as the luminescent substrate.

  Microorganisms include parasites, protozoa, bacteria, fungi, and viruses. Parasites are all those that take the form of infection in tissues and individuals (specifically, animals), such as Anixus, Pseudoterranova, and Trichinella. Protozoa are all those that take infection forms in tissues and individuals (specifically, animals), such as cryptosporidium, neospora, malaria, toxoplasma, isosbola, and cyclosbora. Bacteria are all infectious forms in cells, tissues, and individuals (animals or plants), such as Salmonella typhi, Salmonella, carbon, Bacillus cereus, tuberculosis, and Agrobacterium. Fungi are all infectious forms in cells, tissues and individuals (specifically animals), such as Aspergillus fungus, Mucor lysopus fungus, Penicillium fungus, Acremonium fungus and the like. Viruses are all infectious forms in cells, tissues, and individuals (specifically animals), such as norovirus, tarovirus, and influenza virus.

  The combination of the host and the microorganism is as described above. When the host is an individual or a tissue, the microorganisms are mainly parasites, protozoa, fungi, bacteria, and viruses. When the host is a cell, fungi, bacteria and viruses are mainly used as microorganisms.

Returning to the description of FIG. 1, the objective lens 112 is inverted from below the sample stage 102. An objective lens heater 114 is attached around the objective lens 112, and the objective lens 112 and the objective lens heater 114 are in contact with each other. Here, the present applicant changed the numerical aperture (NA) of the objective lens based on the numerical aperture commonly used in conventional fluorescence imaging microscopes, etc. It has been clarified that a luminescent image can be obtained in a short time (for example, about 1 to 20 minutes) by imaging a luminescent component (see WO 2006/088109). Specifically, the applicant of the present invention has an objective lens for easily imaging bioluminescence with a value of (NA ÷ β) ² represented by a numerical aperture (NA) and a projection magnification (β) of 0. It has been clarified that those having a value of 01 or more are preferred, and those having a value of 0.039 or more are more preferred (see WO 2006/088109). Furthermore, as a result of intensive studies, the applicant has a value of (NA ÷ β) ² as an objective lens for obtaining a luminescent image that can be viewed and analyzed within 5 minutes (in some cases about 1 minute). It has been clarified that 0.071 or more is preferable (see WO 2006/088109). Therefore, the objective lens 112 used in the present embodiment preferably has a value of (NA ÷ β) ² of 0.01 or more, more preferably 0.039 or more, and the value is What is 0.071 or more is further more preferable.

  The objective lens heater 114 is connected to a heater temperature controller 116. In order to maintain the objective lens 112 at a constant temperature from the outside, the temperature is set at intervals of 0.5 ° C. by the heater temperature controller. In addition, an objective lens Z-axis drive mechanism 118 that drives the objective lens along the Z axis (optical axis direction) is provided around the objective lens heater 114. The operation of the objective lens Z-axis drive mechanism 118 is controlled by a computer.

  Below the objective lens 114, a CCD camera 122 as a light detector is placed so that the center of the light receiving surface is aligned with the optical axis. The light emitted from the sample S is detected as a two-dimensional image by the CCD camera 122. Since the light emitted from the sample S is weak light, the CCD camera 122 is used as highly sensitive as possible. The number of pixels of the CCD camera 122 is 1,360 × 1,024. In the present embodiment, the weak light from the sample S can be imaged in a reasonable time (preferably within about 30 minutes) (in other words, the signal detected from the sample S is used to make a reasonable Use a sensitive photodetector that can build an image in time (preferably within about 30 minutes). Specifically, in this embodiment, in order to detect a very weak luminescent component, for example, a high-sensitivity photodetector such as ORCA-AG or ORCA-IIER made by Hamamatsu Photonics, DP30BW made by Olympus, etc. A certain cooled charge coupled photodetecting element is used. In order to detect an extremely bright light-emitting component, a general high-sensitivity video camera (for example, night vision goggles or a silicon multiplier (SIT) camera manufactured by Hamamatsu Photonics) may be used. The CCD camera 122 may be a three-plate color camera so that a color bright field image can be obtained. In the present embodiment, for example, a CMOS image sensor may be used without being limited to the CCD camera.

  In order to suppress the dark current generated from the CCD camera 122, a CCD camera cooling device 124 comprising a Peltier element is installed at the bottom of the CCD camera 122, and the temperature of the CCD camera 122 is cooled and kept at about 0 ° C. An infrared cut filter 120 is disposed above the light receiving surface of the CCD camera 122, and the infrared cut filter blocks infrared light that is not related to signal light as background light. A personal computer 128 including a monitor is connected to the CCD camera 122 through a signal cable. An image of the sample S is drawn on the monitor screen of the personal computer 128.

  The personal computer 128 is sold by, for example, Photometrics or Hamamatsu Photonics as a part of a system including a high-sensitivity photon counting camera such as the CCD camera 122. The personal computer 128 has a function of processing an image captured by the CCD camera 122 (specifically, a light emission image, a fluorescence image, a bright field image, etc.) into a digital file format, and the digital image is stored in the personal computer. It can be operated with various image processing programs set in advance, or can be printed with a printer connected in advance with the personal computer.

  Specifically, the personal computer 128 analyzes the luminescent image captured by the CCD camera 122 to generate information on the ecology of microorganisms contained in the sample S, or the luminescent image captured by the CCD camera 122 and the bright field image, In addition, a superimposed image is created by superimposing the fluorescence image, and the created superimposed image is analyzed to generate information on the ecology of the microorganisms contained in the sample S. Thereby, information on the ecology of microorganisms, such as the behavior and form of microorganisms, localization, and life and death, can be obtained from the luminescence image and the superimposed image. Specifically, the personal computer 128 generates a plurality of information on the ecology of microorganisms contained in the sample S by analyzing a plurality of light emission images repeatedly captured by the CCD camera 122 at predetermined time intervals, A plurality of superimposed images are created by superimposing each light-emitting image repeatedly captured by the camera 122 at predetermined time intervals with each bright-field image and / or each fluorescence image, and the created plurality of superimposed images are analyzed. For example, information on changes in the ecology of microorganisms contained in the sample S is generated. As a result, it is possible to obtain information related to changes in the ecology of microorganisms such as the behavior and morphology of microorganisms, localization, life and death, proliferation, and reduction from time-series emission images and superimposed images. In other words, the behavior, morphology, localization, life / death, proliferation, and decrease of microorganisms can be traced over time from time-series light-emitting images and superimposed images. The predetermined time interval described above may be arbitrarily set, for example, from a short one of several minutes to a long one of several days or weeks.

  Next, an example of main processing performed by the biological sample analyzer 100 will be described with reference to FIG. The experimenter puts the sample S in the sample container C together with the culture solution Cs, puts the sample container C in the water tank T filled with pure water, places the water tank T on the heat plate 104, and puts it in the personal computer 128. When the start of the main process is instructed, the biological sample analyzer 100 executes the following main process with the personal computer 128 as the center. Here, the main process in the case where a light-emitting image, a bright-field image, and / or a fluorescence image are captured over time will be described as an example.

  First, the personal computer 128 instructs the CCD camera 122 to execute imaging, and the predetermined time of the personal computer is stored as the time (imaging time) when the CCD camera 122 captures an image. Store in the area (step SA-1).

  Next, upon receiving an instruction from the personal computer 128, the CCD camera 122 executes imaging of the sample S and acquires a light emission image and a bright field image and / or fluorescence image of the sample S (step SA-2: imaging). Process).

  Next, the CCD camera 122 transfers the light emission image and the bright field image and / or the fluorescence image captured in Step SA-2 to the personal computer 128 (Step SA-3).

  Next, the personal computer 128 acquires the light emission image and the bright field image and / or fluorescence image transferred in step SA-3, and the acquired light emission image and bright field image and / or fluorescence image are acquired in step SA-1. The information is stored in a predetermined storage area of the personal computer in association with the stored imaging time (step SA-4).

  Next, the personal computer 128 creates a superimposed image that is an image in which the light-emitting image stored in step SA-4 and the bright-field image and / or fluorescent image are superimposed (step SA-5).

  Next, the personal computer 128 analyzes the superimposed image created in step SA-5 to generate information on the ecology of the microorganism (step SA-6: analysis step).

  Next, the personal computer 128 uses the superimposed image created in step SA-5, the luminescent image stored in step SA-4, and the bright-field image and / or the information on the ecology of the microorganism generated in step SA-6. A thumbnail image is displayed on the monitor of the personal computer together with the fluorescent image and the imaging time stored in step SA-1 (step SA-7).

  Next, the personal computer 128 measures the elapsed time from the imaging time stored in step SA-1, and when a predetermined time has elapsed from the imaging time (step SA-8: Yes), proceeds to step SA-9. move on.

  Then, the personal computer 128 repeatedly executes the above-described processing centering on the personal computer until a predetermined time elapses from the first imaging time or until a predetermined number of times of imaging is finished (step SA-9: Yes). In this way, information on changes in the ecology of microorganisms is generated.

  As described above in detail, according to the biological sample analyzing apparatus 100, the luminescent image and the bright field image and / or the fluorescent image of the sample S, which is a biological sample including a microorganism having a bioluminescent protein, are captured and captured. Since the ecology of microorganisms is analyzed based on a superimposed image obtained by superimposing a luminescent image and a bright-field image and / or a fluorescence image, microorganisms in a biological sample such as a thick living tissue are identified as the microorganisms. In a state where the biological sample is present without being removed from the biological sample, it can be clearly captured with a simple apparatus configuration without damaging the biological sample. It is possible to analyze (know) accurately and in detail the behavior, morphology, localization, life and death of microorganisms in a biological sample. In other words, by superimposing the luminescent image and the bright field image and / or the fluorescence image, the light signal from the thick biological sample can be measured with high sensitivity without any damage to the tissue without background, As a result, ecology such as the behavior, morphology, localization, life and death of microorganisms in the biological sample can be analyzed accurately and in detail.

  Moreover, according to the biological sample analyzer 100, the luminescent image and the bright field image and / or the fluorescence image are captured over time, and the captured luminescence image and the bright field image and / or the fluorescence image are superimposed. Because changes in the ecology of microorganisms are analyzed based on multiple superimposed images, microorganisms in a biological sample such as a thick living tissue can be present in the biological sample without removing the microorganism from the biological sample. In this state, the biological sample can be clearly captured with a simple apparatus configuration without damaging the biological sample, and as a result, the change in the ecology of microorganisms in the biological sample, specifically the behavior of microorganisms in the biological sample, Localization, life and death, proliferation, reduction, etc. can be analyzed (tracked) accurately and in detail over time. In other words, by observing the image of the light signal in a time-lapse manner, the light signal from a thick biological sample can be measured (tracked) over time with high sensitivity and no background damage. As a result, biological changes such as behavior, localization, life / death, proliferation, and decrease of microorganisms in a biological sample can be analyzed accurately and in detail over time.

In addition, according to the biological sample analyzer 100, a luminescent image, a bright-field image, and a fluorescent image are picked up by the cooling charge coupled photodetecting element, so that a highly sensitive image can be obtained. It is possible to analyze the ecology and changes of microorganisms more accurately and in detail. Moreover, according to the biological sample analyzer 100, the value of (NA ÷ β) ² represented by the numerical aperture (NA) and the projection magnification (β) is 0.01 or more, preferably 0.039 or more, more preferably. Since a luminescent image, bright field image, and fluorescent image are captured using an objective lens that is 0.071 or more, a highly sensitive image can be obtained. More accurate and detailed analysis can be performed.

  In addition, the experimenter administers the bioluminescent protein to the microorganism by various methods, further administers the microorganism to an individual such as an animal, adds a luminescent substrate to the individual, and then uses the biological sample analyzer 100 to Luminescence emitted from microorganisms present in the individual or in the tissue of the individual may be imaged.

  In the present embodiment, the measurement of the luminescent signal may last for several tens of minutes (specifically, the exposure time during imaging is several tens of minutes). It is desirable to fix such a sample. In addition, it is desirable to use a cooled charge coupled photodetection element capable of detecting very low level light for imaging of the luminescent signal component.

In Example 1, luminescence observation and bright field observation were performed using a fragment of the small intestine of a mouse as a sample.
(1) Preparation of sample First, a luciferase gene constant expression vector was introduced into Cryptosporidium by electroporation. Then, mice were orally infected with luciferase-expressing Cryptosporidium in the oocyst form, and Cryptosporidium was proliferated in the intestine. Then, a fragment of the mouse small intestine was collected, and the collected fragment was placed in a 35 mm cover glass dish containing PBS (phosphate buffered saline) containing 1 mM luciferin.
(2) Observation of sample In Example 1, the fragment | piece in a cover glass dish was observed using the light emission imaging system LUMINOVIEW LV100 (made by Olympus). The objective lens used in Example 1 is for a commercially available microscope and has a specification of 20 × NA 0.75. The total magnification corresponding to the magnification Mg is 4 times. The CCD camera used in Example 1 is a digital camera ORCA-AG (manufactured by Hamamatsu Photonics) for a microscope cooled at −30 ° C., the CCD element as the CCD 3 is a 2/3 inch type, and the number of pixels is 1,360. × 1,024, pixel size is 6.45 μm square. In Example 1, an image of a specimen by self-luminescence was exposed for 1 minute and imaged. The sample was observed in an environment of 37 ° C. because the sample (specimen) was placed in the incubator.
(3) Observation Results As shown in FIG. 4, it was possible to confirm that Cryptosporidium was infected in the oocyst form in the small intestine of mice. FIG. 4 is a diagram illustrating an example of a bright-field image, a luminescent image, and a superimposed image when a mouse small intestine fragment is imaged.

  In Example 2, the growth of Cryptosporidium in the mouse intestine was observed by performing luminescence observation and bright field observation over time using a fragment of the mouse small intestine as a sample. The adjustment of Cryptosporidium, the adjustment of the mouse, the luminescence imaging system, the objective lens, the CCD camera, and the imaging conditions are the same as those in Example 1. In Example 2, the imaging interval was 30 minutes, and 24-hour time-lapse imaging was performed. As a result, it was possible to capture how Cryptosporidium proliferated over time.

  As described above, the biological sample analysis method according to the present invention can be suitably used in fields such as medical treatment, biotechnology, and food analysis.

1 is a diagram illustrating a configuration of a biological sample analyzer 100. FIG. 1 is a diagram illustrating a configuration of a biological sample analyzer 100. FIG. It is a flowchart which shows an example of the main process which the biological sample analyzer 100 performs. It is a figure which shows an example of the bright field image, the light emission image, and a superimposition image when the small intestine fragment | piece of a mouse | mouth is imaged.

Explanation of symbols

100 Biological sample analyzer
112 Objective lens
122 CCD camera
124 CCD camera cooling device
128 Personal computer

Claims (5)

By selectively detecting the faint light emitted from the microorganism among a plurality of biological samples including a microorganism that is a parasite or a protozoan having a bioluminescent protein, which is a host that is a thick living tissue, An imaging step of repeatedly capturing a luminescence image of the microorganism and repeatedly capturing a bright field image of the biological sample and / or a fluorescent image of the biological sample ;
Analysis for analyzing ecology of the microorganisms in said plurality of biological samples based on which the superimposed image is an image obtained by superimposing said bright field image and / or the fluorescence image and the previous SL luminescent image taken by the image pickup step Process,
A biological sample analysis method comprising: The biological sample analysis method according to claim 1 , wherein in the imaging step, at least the emission image is captured by a charge-coupled light detection element. In the imaging step, at least the emission image is captured using an objective lens having a value of (NA ÷ β) ² expressed by a numerical aperture (NA) and a projection magnification (β) of 0.01 or more. The biological sample analysis method according to claim 1 or 2 , characterized in that The biological sample analysis method according to claim 3 , wherein the value of the (NA ÷ β) ² of the objective lens is 0.039 or more. The biological sample analysis method according to claim 4 , wherein the value of the (NA ÷ β) ² of the objective lens is 0.071 or more.