JP5058483B2 - Long-term or continuous detection method for biological samples - Google Patents

Description

  The present invention relates to a method for detecting a biological activity in a biological sample such as a cell or tissue for a long period or continuously without losing the activity as much as possible. The invention also includes software for an automated device executed by the method.

  In research in the fields of biology and medicine, techniques for detecting the biological activity of biological samples such as cells by reporter assays have been widely used. Reporter assays can be used to visualize biological activities that cannot be visually examined. In conventional clinical tests, only biological substances (nucleic acid, blood, hormones, proteins, etc.) to be examined from biological samples are isolated by various separation methods, and the amount and activity of the isolated biological substances are determined as reagents. And reacted. However, in living organisms, the interaction between various biological materials shows true biological activity. In recent years, when researching or developing medical drugs, drugs that most effectively act on biological activity in living biological samples have become critical conditions. In reporter assays for living biological samples, there is a growing need to image biological samples and biological substances to be examined and observe dynamic changes in and out of biological samples over time. .

Specifically, in research fields that utilize observation using luminescence (bioluminescence, chemiluminescence) or fluorescence as a reporter substance, time-lapse or video imaging is required to capture the dynamic functional expression of protein molecules in a sample. It has been. At present, dynamic changes are observed using an image picked up from a fluorescent sample (for example, observation of a moving image of one protein molecule using fluorescence). In the case of imaging a fluorescent sample, the amount of light emitted from the fluorescent sample decreases with the lapse of time by continuing to irradiate excitation light, so that a stable image that can be used for quantitative evaluation can be taken over time. However, it was possible to take a clear, high spatial resolution image with a short exposure time. On the other hand, in the time-dependent observation of dynamic changes by images for a luminescent sample, the luminescence from the luminescent sample is extremely small, so a CCD camera equipped with an image intensifier was used to observe the luminescent sample. It was done. In the case of imaging of a luminescent sample, it was not necessary to irradiate excitation light, so that stable images that could be used for quantitative evaluation could be taken over time.

Until now, in the observation of a luminescent sample, the amount of luminescence from the luminescent sample has been measured. For example, in the observation of a cell into which a luciferase gene has been introduced, the amount of luminescence from the cell due to the luciferase activity was measured in order to investigate the intensity of the luciferase gene expression (specifically, the expression level). . The amount of luminescence from the cells due to luciferase activity is measured by first reacting a cell lysate in which cells are lysed with a substrate solution containing luciferin, ATP, magnesium, etc., and then reacting with the substrate solution. The amount of luminescence from was quantified with a luminometer using a photomultiplier tube. That is, the amount of luminescence was measured after cell lysis. Thereby, the expression level of the luciferase gene at a certain time point could be measured as an average value of the whole cell. Here, methods for introducing a luminescent gene such as a luciferase gene into a cell as a reporter gene include, for example, a calcium phosphate method, a lipofectin method, an electroporation method, and the like, and each method is properly used depending on the purpose and the type of cell. Yes. In addition, when investigating the intensity of luciferase gene expression in cells transfected with a luciferase gene as a reporter gene using the amount of luminescence from the cell due to luciferase activity as an indicator, the target luciferase gene is introduced upstream or downstream of the luciferase gene introduced into the cell. By linking DNA fragments, it is possible to examine the effect of the DNA fragment on the transcription of the luciferase gene. In addition, genes such as transcription factors that are thought to affect the transcription of the luciferase gene to be introduced into cells are linked to the expression vector. By co-expressing with the luciferase gene, the effect of the gene product of the gene on the expression of the luciferase gene can be examined.

In addition, in order to capture the expression level of the luminescent gene over time, it is necessary to measure the amount of luminescence from living cells over time. In order to measure the amount of luminescence from living cells over time, first add the function of a luminometer to the incubator that cultivates the cells, and then quantitate the amount of luminescence from the whole cell population cultured at regular intervals with the luminometer. The procedure was to do. As a result, it was possible to measure an expression rhythm with a certain periodicity, and thus, it was possible to capture changes over time in the expression level of the luminescent gene in the entire cell. On the other hand, when the expression of the luminescent gene is transient, the expression level in individual cells varies greatly. For example, even in the case of cloned cultured cells such as HeLa cells, the response of drugs via receptors on the cell membrane surface may vary among individual cells. That is, several cells may be responding even if the response as a whole cell is not detected. For this reason, when the expression of the luminescent gene is transient, it is important to measure the amount of luminescence from individual cells over time rather than from the whole cell . The time-dependent measurement of the amount of light emitted from living individual cells using a microscope shows that the light emitted from each cell is extremely weak, so it can be exposed for a long time with a cooled CCD camera at a liquid nitrogen temperature level, or an image intensifier. Or a photon counting device. As a result, it was possible to capture changes over time in the expression level of the luminescent gene in individual living cells.

However, when imaging a weakly luminescent sample, the amount of light emitted from the luminescent sample is extremely small, so it cannot be viewed with the naked eye, and the amount of light must be accumulated using a storage-type imaging means such as a CCD. Therefore, there is a restriction that an image cannot be generated. Moreover, in the cell group constituting a single cell or tissue, the weak light generated from each cell is too weak, so that the exposure time required for taking a clear image becomes long. there were. That is , since the imaging time interval is limited by the amount of light per unit time, when imaging a luminescent sample with weak light emission, a clear image can be taken over time at a long time interval, for example, at an interval of 60 minutes. Even if it was possible, there was a problem that imaging could not be performed in real time with a short exposure time of about 10 to 30 minutes , and thus with an exposure of 1 to 5 minutes . In particular, when living cells are exposed for a long time (for example, 50 minutes or longer), the cells themselves may move even on a support such as a culture vessel and a clear image may not be formed. Generally, in order to perform an analysis using an image, it is necessary to recognize an accurate contour. Therefore, the analysis result may be inaccurate when the image is unclear.

The present invention has been made in view of the above-described problems, and provides a biological sample detection device capable of capturing an image so as not to cause a time difference even with a biological sample including many cells that emit faint light. With the goal.

  In order to solve the above-described problems and achieve the object, the biological sample detection device according to claim 1 according to the present invention maintains a high numerical aperture (NA) objective lens and a biological sample in a living and light-shielding environment. And a sample holding unit, and imaging means for acquiring imaging data in a time corresponding to a desired phenomenon or activity in the biological sample in cooperation with the objective lens. Here, it is preferable that the imaging unit further includes an interval condition setting unit, and acquires a plurality of images related to the biological sample at different times. The present invention also includes a detection method using the above apparatus. Here, in the said method, the image analysis containing a cell is also attained by including the cell which generate | occur | produces weak light at least.

  Note that a luminescent sample imaging method that embodies the above-described apparatus and method is a luminescent sample imaging method for imaging a luminescent sample, which is expressed by a numerical aperture (NA) and a projection magnification (β) of (NA ÷ β) 2 An objective lens having a power value of 0.01 or more, preferably 0.039 or more is used. In addition, the present invention can be embodied in a luminescent cell imaging method, and is expressed by a numerical aperture (NA) and a projection magnification (β) in a luminescent cell imaging method for imaging a luminescent cell into which a luciferase gene has been introduced (NA). An objective lens having a square value of ÷ β) of 0.01 or more, preferably 0.039 or more is used.

  Further, the present invention relates to an objective lens, and the objective lens according to claim 9 according to the present invention is an objective lens used in a luminescent sample imaging method for imaging a luminescent sample, wherein the numerical aperture (NA) and the projection magnification ( The square value of (NA ÷ β) represented by β) is 0.01 or more, preferably 0.039 or more.

  The present invention also relates to an objective lens, and the objective lens according to claim 16 according to the present invention is an objective lens used in a luminescent cell imaging method for imaging luminescent cells into which a luciferase gene has been introduced. A square value of (NA ÷ β) represented by a numerical aperture (NA) and a projection magnification (β) is 0.01 or more, preferably 0.039 or more.

  In addition, the present invention relates to an objective lens, and the objective lens according to claim 17 according to the present invention is an objective lens used in a luminescent sample imaging method for imaging a luminescent sample. A square value of (NA ÷ β) represented by a numerical aperture (NA) and a projection magnification (β) is described on a packaging container for packaging the above.

  In the luminescent sample imaging method according to the present invention, the square value of (NA ÷ β) represented by the numerical aperture (NA) and the projection magnification (β) is 0.01 or more, preferably 0.01 or more, preferably An objective lens that is 0.039 or more is used. As a result, a luminescent sample (for example, a luminescent protein (for example, a luminescent protein expressed from an introduced gene (for example, luciferase gene)), a luminescent cell, or a collection of luminescent cells, Even in the case of sex tissue samples and luminescent individuals (such as animals and organs), a clear image can be taken with a short exposure time and in real time.

  In the luminescent cell imaging method according to the present invention, the square value of (NA ÷ β) represented by the numerical aperture (NA) and the projection magnification (β) is 0.01 or more, preferably 0.039 or more. Since an objective lens is used, it is possible to take a luminescent cell into which a luciferase gene has been introduced as an imaging target, and to take a clear image with a short exposure time and in real time.

  In the objective lens according to the present invention, the square value of (NA ÷ β) expressed by the numerical aperture (NA) and the projection magnification (β) is 0.01 or more, preferably 0.039 or more. Luminescent samples with low luminescence (eg, luminescent proteins (eg, luminescent proteins expressed from introduced genes (eg, luciferase genes)), luminescent cells or aggregates of luminescent cells, Even in the case of a tissue sample or a luminescent individual (such as an animal or an organ), a clear image can be taken with a short exposure time and in real time. Specifically, there is an effect that a luminescent cell into which a luciferase gene is introduced can be imaged and a clear image can be taken with a short exposure time and in real time.

  In addition, the objective lens according to the present invention has both a large numerical aperture and a small magnification as compared with the conventional objective lens. Therefore, if the objective lens according to the present invention is used, a wide range of resolution can be achieved. It can capture well. Thereby, for example, a moving luminescent sample or a luminescent sample distributed over a wide range can be an imaging target. In addition, the objective lens according to the present invention is expressed by the numerical aperture (NA) and the projection magnification (β) (NA ÷ β) on the objective lens and / or a packaging container (package) for packaging the objective lens. A square value (for example, 0.01 or more, preferably 0.039 or more) was expressed. Thus, for example, if a person who observes a luminescent image confirms the square value of (NA ÷ β), an objective lens suitable for imaging a luminescent sample with a short exposure time and in real time can be obtained. There is an effect that it can be easily selected.

  Hereinafter, embodiments of a luminescent sample imaging method, a luminescent cell imaging method, and an objective lens according to the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

First Embodiment A configuration of an apparatus for carrying out a luminescent sample imaging method according to the present invention will be described with reference to FIG. FIG. 1 is a diagram illustrating an example of a configuration related to an apparatus according to the first embodiment of the present invention. As shown in FIG. 1, an apparatus for carrying out a luminescent sample imaging method according to the present invention is for imaging a sample 1 to be imaged with a short exposure time, and in real time. The condenser lens 3, the CCD camera 4, and the monitor 5 are included. The apparatus may further include a zoom lens 6 as shown.

  Sample 1 is a luminescent sample, for example, a luminescent protein (for example, a luminescent protein expressed from an introduced gene (such as a luciferase gene)), a luminescent cell, an aggregate of luminescent cells, or a luminescent property. Tissue samples, luminescent organs, and luminescent individuals (animals, etc.). Sample 1 may specifically be a luminescent cell into which a luciferase gene has been introduced. The objective lens 2 has a square value of (NA ÷ β) expressed by a numerical aperture (NA) and a projection magnification (β) of 0.01 or more, preferably 0.039 or more. The condenser lens 3 collects light emitted from the sample 1 that has reached through the objective lens 2. The CCD camera 4 is a cooled CCD camera at about 0 ° C. and images the sample 1 through the objective lens 2 and the condenser lens 3. The monitor 5 outputs an image captured by the CCD camera 4.

  Then, the square of (NA / β) is written on the objective lens 2 and the packaging container (package) of the objective lens 2. Here, an example of an objective lens in which a square value of (NA / β) is described will be described with reference to FIG. FIG. 2 is a diagram illustrating an example of the objective lens 2 in which a square value of (NA / β) is expressed. Conventional objective lenses include lens type (eg “PlanApo”), magnification / NA oil attack (eg “100 × / 1.40 oil”) and infinity / cover glass thickness (eg “∞ / 0.17”). It was written. However, the objective lens according to the present invention (objective lens 2) includes a lens type (for example, “PlanApo”), magnification / NA oil immersion (for example, “100 × / 1.40 oil”), infinity / cover glass thickness (for example, In addition to “∞ / 0.17”), an injection opening angle (for example, “(NA / β) squared: 0.05”) is also written.

  As described above, in the apparatus for carrying out the luminescent sample imaging method according to the present invention, the objective lens 2 is represented by 2 (NA ÷ β) represented by the numerical aperture (NA) and the projection magnification (β). The power value is 0.01 or more, preferably 0.039 or more. As a result, a luminescent sample (for example, a luminescent protein (for example, a luminescent protein expressed from an introduced gene (for example, luciferase gene)), a luminescent cell, or a collection of luminescent cells, A clear image can be taken with a short exposure time and even in real time even with a sex tissue sample or a luminescent individual (such as an animal or an organ). Specifically, a luminescent cell into which a luciferase gene is introduced can be imaged, and a clear image can be taken with a short exposure time and in real time. In addition, since the objective lens 2 has a larger numerical aperture and a lower magnification as compared with a conventional objective lens, a wide range can be imaged with high resolution by using the objective lens 2. Thereby, for example, a moving luminescent sample, a moving luminescent sample, or a luminescent sample distributed over a wide range can be set as an imaging target. In addition, the objective lens 2 is expressed by a numerical aperture (NA) and a projection magnification (β) of 2 (NA ÷ β) on the objective lens 2 and / or a packaging container (package) that wraps the objective lens 2. A power value (for example, 0.01 or more, preferably 0.039 or more) was expressed. Thus, for example, if a person who observes a luminescent image confirms the square value of (NA ÷ β), an objective lens suitable for imaging a luminescent sample with a short exposure time and in real time can be obtained. Easy to choose.

  Conventional reporter assays using the luciferase gene measure the amount of luminescence after lysing the cells, so only the expression level at a certain point in time can be captured, and the average value of the entire cell is measured. . Further, in measurement while culturing, changes in the expression level of cell colonies over time can be caught, but changes in the expression level in individual cells cannot be caught. In order to observe the luminescence of individual cells with a microscope, the amount of luminescence from living cells is extremely weak, so it can be exposed for a long time with a cooled CCD camera at the liquid nitrogen temperature level, or an image intensifier is attached. You have to do photon counting with a CCD camera. Therefore, the camera for detecting light emission is expensive and large. However, when observing the luminescence of individual cells exhibiting luciferase activity as a reporter gene product with a microscope, an image intensifier is attached if the apparatus for carrying out the luminescent sample imaging method according to the present invention is used. The quantitative image can be acquired using a cooled CCD camera at about 0 ° C. That is, if the apparatus for carrying out the luminescent sample imaging method according to the present invention is used, the luminescence of individual cells can be observed with a cooled CCD camera at about 0 ° C. in an alive state. No fire or photon counting device is required. That is, the luminescent sample can be imaged at low cost. In addition, if the apparatus for performing the luminescent sample imaging method according to the present invention is used, the luminescence of individual living cells can be observed over time while being cultured, and can also be observed in real time. . Moreover, if the apparatus for performing the luminescent sample imaging method according to the present invention is used, it is possible to monitor the response of drugs and stimuli under different conditions for the same cell.

Here, in order to facilitate understanding of the luminescent sample imaging method, the luminescent cell imaging method and the objective lens according to the present invention, a conventional objective lens and luminescent image observation using the objective lens will be briefly described.
In general, the spatial resolution ε in microscopic observation is expressed by the following mathematical formula 1.
ε = 0.61 × λ ÷ NA (Formula 1)
(In Equation 1, λ is the wavelength of light and NA is the numerical aperture.)
Further, the diameter d of the observation range is expressed by the following mathematical formula 2.
d = D ÷ M (Formula 2)
(In Equation 2, D is the number of fields of view and M is the magnification. The number of fields of view is generally 22 to 26.)
Conventionally, the focal length of an objective lens for a microscope has been set to 45 mm according to international standards. Recently, an objective lens having a focal length of 60 mm has been used. If a lens with a large NA, that is, a high spatial resolution is designed on the assumption of this focal length, the working distance (WD) is generally about 0.5 mm, and even a long WD design is about 8 mm. When such an objective lens is used, the observation range is about 0.5 mm in diameter.

  However, when observing a cell group, tissue, or individual dispersed in a dish or glass bottom dish, the observation range may range from 1 to several centimeters. When it is desired to observe such a range with high resolution, the NA must be maintained at a large value while the magnification is low. In other words, since NA is the ratio of the lens radius to the focal length, an objective lens that can observe a wide range with a large NA needs to be low magnification. As a result, such an objective lens has a large aperture. In manufacturing a large-diameter objective lens, generally high accuracy is required in terms of uniformity of physical properties and coating uniformity of an optical material and lens shape.

In the case of microscopic observation, the light transmittance of the optical system, the numerical aperture of the objective lens, the projection magnification on the chip surface of the CCD camera, the performance of the CCD camera, etc. greatly affect the brightness of the image. The brightness of the image is evaluated by the square of the value obtained by dividing the numerical aperture (NA) by the projection magnification (β), that is, the square of (NA / β). Here, as shown in FIG. 3, the objective lens generally has a relationship of the following Equation 3 between the incident aperture angle NA and the exit aperture angle NA ′, where NA ′ 2 is the observer's eyes or CCD camera. It is a value indicating the brightness that reaches.
NA ′ = NA ÷ β (Formula 3)
(In Equation 3, NA is the incident aperture angle (numerical aperture), NA ′ is the exit aperture angle, and β is the projection magnification.)
In a general objective lens, NA ′ is at most 0.04, and NA ′ 2 is 0.0016. Further, when the square value of the image brightness (NA / β) in an objective lens of a general microscope currently on the market is examined, as shown in FIG. 4, the range is from 0.0005 to 0.002. Met.

  However, using a microscope equipped with a commercially available objective lens shown in FIG. 4, for example, even when observing a luminescent cell expressing a luciferase gene in a cell, the luminescence from the cell is visually observed. In addition, even if a light emission image taken using a CCD camera cooled to about 0 ° C. is observed, light emission from the cells cannot be confirmed. When observing a luminescent sample, it is not necessary to project excitation light necessary for fluorescence observation. For example, in epifluorescence observation, the objective lens satisfies both functions of an excitation light projection lens and a lens that collects fluorescence to form an image. Therefore, in order to observe light emission with a small amount of light with an image, an objective lens having large NA and small β characteristics is required. As a result, the objective lens tends to have a large aperture. Note that such an objective lens is required to be easily designed and manufactured by simplifying the function without considering the function of projecting the excitation light.

In the research field using luminescence and fluorescence observation, time-lapse and moving image capturing are required to capture the dynamic functional expression of protein molecules in a sample. Recently, video observation of one molecule of protein using fluorescence has been performed. In these imaging operations, the exposure time per image frame becomes shorter as the number of imaging frames per unit time increases. Such observation requires a bright optical system, particularly a bright objective lens. However, since the amount of photoprotein is less than that of fluorescence, it often takes an exposure time of, for example, 20 minutes to image one frame. In order to perform time- lapse observation with such an exposure time, a dynamic change is limited to a very slow sample. For example, in cells that divide once every hour, changes within that cycle cannot be observed. Therefore, it is important to improve the brightness of the optical system in order to efficiently image a small amount of light while maintaining a high signal-to-noise ratio.

The objective lens of the present invention manufactured based on the above circumstances has characteristics of large NA and small β as compared with a commercially available objective lens (see, for example, FIG. 4). That is, as will be described later, when a low-temperature cooled CCD of 0 ° C. to 5 ° C. is used, each square value of (NA / β) is in the range of 0.01 to 0.09. Luminescence images of cells by photoproteins could be generated within 5 minutes, and the amount of luminescence for individual cells could be measured. On the other hand, under the same conditions, when the square value of (NA / β) was 0.007 or less, a luminescent image that could be recognized by the naked eye or image analysis software could not be generated. Therefore, the square value (or NA′2) of (NA / β) of the objective lens capable of generating a luminescent image in the present invention is a value significantly larger than the range conventionally used. In other words, it can be said that the objective lens of the present invention is a bright objective lens under conditions different from those conventionally used . Thereby, if a bright objective lens like the objective lens of this invention is used, the light emission from the light emission sample with little light quantity can be observed with an image. In order to observe a darker image, a CCD camera cooled to about 0 ° C. without mounting an image intensifier by mounting the objective lens of the present invention having a large numerical aperture on a stereomicroscope, Cell luminescence can be observed with images. In addition, there is a method of increasing the sensitivity with a CCD camera using liquid nitrogen cooling. In this case, the CCD camera becomes very expensive and large-scale. However, if the objective lens of the present invention is used, the light emission of the cells can be observed as an image even with a CCD camera using Peltier cooling.

The objective lens of the present invention has a large aperture of about 5 cm to 10 cm. As a result, a luminescent sample having movements such as shaking, deformation, splitting, and movement, which could not be an imaging target in the past, or a luminescent sample distributed over a wide range can be set as an imaging target. According to the present invention, for example, in a culture sample (a tissue or a group of cells) containing cells, a visual field range equivalent to 1 cm square, preferably 2 cm to 5 cm square or more can be observed, so that various important tissues or organs (for example, The brain, suprachiasmatic nucleus, pancreas, tumor tissue, nematode, etc.) or all of them are preferable in that they can be observed and analyzed in a wide field of view with thin sections and the like as appropriate. In the above description, application of a cryogenic cooling CCD such as liquid nitrogen to the present invention is not excluded. This is because, according to the present invention, high-speed imaging that cannot be obtained even with a cryogenic cooled CCD is realized only by the optical configuration before light reception including the objective lens. Therefore, by combining the method and apparatus of the present invention with a cryogenic cooling CCD, high sensitivity can be achieved and the S / N ratio can be increased, so that the image quality can be improved.

Second Embodiment Next, images actually captured by the weak light sample imaging unit and the weak light sample imaging device according to the second embodiment are shown in FIGS. The images shown in FIGS. 5-1 to 5-4 show that the luciferase gene “pGL3-control vector (manufactured by Promega)” was introduced into human-derived HeLa cells, cultured for one day, and then washed with Hanks balanced salt solution. It is the image which imaged the sample produced by substituting with the Hanks salt solution containing 1 mM luciferin.

The lenses used as the objective lens 2 and the condenser lens 3 for image formation are commercially available objective lenses for microscopes having specifications of “Oil, 40 times, NA 1.0” and “5 times, NA 0.13”, respectively. The total magnification corresponding to the magnification Mg of the imaging optical system is 8 times. The CCD camera 4 used is a cooled CCD camera for astronomical observation cooled by 0 ° C. (manufactured by SBIG). The CCD element is a 2/3 inch type, the number of pixels is 765 × 510, and the pixel size is 9 μm square.

FIG. 5A is an image obtained by capturing a bright field image of the specimen, and FIGS. 5B and 5C are images obtained by capturing images of the specimen by self-emission, which are exposed for 1 minute and 5 minutes, respectively. This is a captured image. FIG. 5-4 is an image obtained by superimposing and displaying the two images shown in FIGS.
As shown in FIG. 5B, according to the feeble light sample imaging unit and the feeble light sample imaging device according to the second embodiment, even with a relatively high temperature cooled CCD cooled at 0 ° C., photon counting is performed. Without a short exposure time of 1 minute, the weak light emitted by the luciferase gene can be imaged. Further, as shown in FIG. 7-3, by setting the exposure time to 5 minutes, it is possible to image a luciferase gene that emits weaker light. Furthermore, as shown in FIG. 5-1, it is possible to capture a bright field image of a specimen. As shown in FIG. 5-4, light emission is achieved by superimposing a bright field image and a self-luminous image. In addition to observing the position of the luciferase gene in the specimen, cells containing the luciferase gene that emit light can be identified. Non-Patent Document 1 (David K. Welsh, Seung-Hee Yoo, Andrew C. Liu, Joseph S. Takahashi, and Steve A. Kay: "Bioluminescence Imaging of Individual Fibroblasts Reveals Persistent, Independently Phased Circadian Rhythms of Clock Gene Expression , "Current Biology, Vol. 14 (2004) 2289-2295.), As shown in Figure 2 of this document, it is very difficult to identify the cells in which the specimen is imaged in a mosaic pattern and emit light. Met.

  Furthermore, an example of a reporter assay over time with a single cell will be described as another example of experimental results performed by the weak light specimen imaging unit and the weak light specimen imaging device according to the second embodiment.

  In this reporter assay, first, a vector “pcDNA6 / TR (manufactured by Invitrogen)” that constantly expresses tetracycline repressor (TetR) and an expression vector “pcDNA4 / TO (Invitrogen) having a tetracycline operator (TetO2)” are used. And a luciferase gene-linked plasmid are co-expressed in HeLa cells to prepare a specimen. In this state, as shown in FIG. 6A, since the TetR homodimer is bound to the TetO2 region, transcription of the luciferase gene is suppressed. Next, as shown in FIG. 6-2, tetracycline is added to the culture solution to bind to the TetR homodimer, and by changing the three-dimensional structure of the TetR homodimer, the TetR homodimer is separated from TetO2, and the transcription of the luciferase gene is performed. To induce. The culture solution is a D-MEM medium containing 10 mM HEPES and contains 1 mM luciferin.

  The lenses used as the objective lens 2 and the condenser lens 3 for image formation are commercially available objective lenses for microscopes having specifications of “Oil, 20 times, NA 0.8” and “5 times, NA 0.13”, respectively. The total magnification corresponding to the magnification Mg is 4 times. The CCD camera 4 used was a digital camera “DP30BW (manufactured by Olympus)” cooled at 5 ° C., and the CCD element was a 2/3 inch type, the number of pixels was 1360 × 1024, and the pixel size was 6 μm square.

  FIG. 7-1 is an image obtained by capturing a bright-field image of the specimen before adding tetracycline. FIG. 7-2 is an image obtained by exposing an image by self-emission of a specimen 9 hours after the addition of tetracycline for 1 minute. FIG. 7C is an image obtained by superimposing the two images shown in FIGS. 7A and 7B, and is an image partially enlarged. These observations are performed at room temperature (25 ° C.). When the specimen is placed in the incubator, or when part or all of the imaging unit or imaging apparatus is also stored in the incubator, observation can be performed in an environment of 37 ° C.

  As shown in FIG. 7-2, according to the weak light sample imaging unit and the weak light sample imaging device according to the second embodiment, the cooling CCD cooled at 5 ° C. has a short time of 1 minute without photon counting. The weak light emitted by the luciferase gene can be imaged with the exposure time.

Further, as shown in FIG. 7-3, by superimposing the bright-field image and the self-luminous image, the position of the luciferase gene that emits light can be observed with a clear image, and the cell containing this luciferase gene Can be easily identified.

  FIG. 8 is a diagram showing the results of measuring the change over time in the luminescence intensity of the luciferase gene in the regions ROI-1 and ROI-2 shown in FIG. 7-3. FIG. 8 shows that luminescence was captured 2 hours after the addition of tetracycline and reached a plateau 6-7 hours later. As described above, according to the weak light sample imaging unit and the weak light sample imaging device according to the second embodiment, the position of the luciferase gene that emits light is identified and tracked in time series, and the temporal change of the luminescence phenomenon is measured. can do.

  By the way, in the weak light sample imaging unit and the weak light sample imaging device according to the second embodiment, the imaging optical system including the objective lens 2 and the focusing lens 3 for imaging is an infinity correction system. Thus, various optical elements are arranged between the objective lens 2 and the imaging lens 3, and the sample 1 can be observed by various methods.

  FIG. 9 is a schematic diagram showing a partial configuration in the case where the weak light sample imaging apparatus according to the second embodiment is provided with an epi-illumination apparatus. As shown in FIG. 9, the epifluorescent apparatus includes a fluorescent cube 24 as a fluorescent unit, a fluorescent light projecting tube 25, and an excitation light source 26. The fluorescence cube 24 is an excitation filter 21 as an excitation light transmission filter that selectively transmits excitation light for exciting the specimen 1 and fluorescence that selectively transmits fluorescence emitted from the specimen 1 excited by the excitation light. An absorption filter 22 as a transmission filter and a dichroic mirror 23 that reflects excitation light and transmits fluorescence are integrally provided. The fluorescent light projecting tube 25 includes a condensing lens 25a, a brightness stop making lens 25b, a light projecting lens 25c, a brightness stop AS, and a field stop FS coaxially.

  The excitation light source 26 is a light source that emits excitation light, and is realized by a mercury lamp, a xenon lamp, a laser, or the like. The excitation light source 26 and the fluorescent light projecting tube 25 as the fluorescence irradiation means reflect the excitation light by the dichroic mirror 23 and irradiate the specimen 1. The excitation filter 21 is a band pass filter that extracts excitation light from the light emitted from the excitation light source 26, and the absorption filter 22 is a long wave pass filter having a predetermined cutoff wavelength. The fluorescent cube 24 is detachably disposed on the optical axis OA2 between the objective lens 60 and the imaging lens 70. In addition, together with the fluorescent cube 24, a plurality of replacement fluorescent cubes having different optical characteristics with respect to at least one of excitation light and fluorescence are integrally held, and one fluorescent cube among the held fluorescent cubes is selectively connected to the objective lens 6. A fluorescent cube switching device disposed between the image lens 7 and the image lens 7 may be provided.

  As shown in FIG. 9, by adding an epi-fluorescence device, the weak light sample imaging device according to the second embodiment can capture a fluorescent image of the sample 1. In addition, according to this faint light specimen imaging unit, it is possible to observe faint fluorescence, so there is no need to irradiate the specimen with powerful excitation light as in the case of using an epifluorescence laser scanning confocal microscope, Specimen damage can be reduced. Furthermore, a powerful excitation light source laser, a scanning device such as a galvanometer mirror, a confocal optical system, a photomultiplier, a processing device for image creation, and the like are not required.

Further, the weak light specimen imaging device shown in FIG. 9 can be used as a DNA chip reader. A DNA chip is a resin substrate such as glass or polystyrene that is coated with several hundreds of DNA fragments and synthetic oligonucleotides with a diameter of about 0.3 mm at intervals of about 0.6 mm. It is used to check the existence of specific genes. Usually, the fluorescence emitted from this DNA chip is weak. If the weak light imaging apparatus of the present invention is used, real-time detection is possible even if the amount of solid phase immobilization on a microbiochip in which a conventional genetic or immunological substance is solid phased is greatly reduced. Excellent in terms of

  A general DNA chip reader is a combination of a laser irradiation confocal optical system and a high-speed scanning stage, and the photometry in this apparatus is the sum of the amount of light emitted from the spot excitation light irradiation point. When it changes, the measured light quantity also changes each time, and the absolute light quantity cannot be measured. When the weak light specimen imaging device shown in FIG. 9 is used, for example, the stage holding the DNA chip is moved in a 0.6 mm step with respect to an objective lens having a field of view of 0.5 mm, and photometry is performed every time it is stopped. By doing so, the absolute light quantity can be measured.

  FIG. 10 is a schematic diagram showing a partial configuration in the case where the feeble light sample imaging device according to the second embodiment is provided with a spectrophotometric filter unit. As shown in FIG. 10, the spectrophotometric filter unit 31 has wavelength extraction filters 32 and 33 in the holes 31 a and 31 b, respectively, and is disposed between the objective lens 6 and the imaging lens 7. The filter unit 31 is slidably disposed in a plane substantially orthogonal to the optical axis OA2, and one of the wavelength extraction filters 32 and 33 and the air holes 31c is selectively disposed on the optical axis OA2. The filter unit 31 may be movable in a turret manner, and may hold more wavelength extraction filters.

  For example, when performing spectrophotometry using a multicolor luciferase that emits two colors of light as a specimen 1 using the filter unit 31, the wavelength extraction filter 32 is a long wave pass filter that transmits light having a long wavelength of 650 nm or more. The wavelength extraction filter 33 is preferably a short wave path filter that transmits light having a short wavelength of 550 nm or less. Here, the emission characteristics of multicolor luciferase are represented by spectral characteristic curves 35G and 35R shown in FIG. 11, and the transmittance characteristics of the wavelength extraction filters 32 and 33 are represented by transmittance curves 32LP and 33SP shown in FIG. 11, respectively. Is done.

  In this case, by arranging the wavelength extraction filter 32 on the optical axis OA2 by the filter unit 31 of FIG. 10, the red light emitted from the multicolor luciferase can be measured, and the wavelength extraction filter 33 is arranged on the optical axis OA2. By disposing in this way, the green light emitted from the multicolor luciferase can be measured. Further, the bright field image of the specimen 1 can be observed by arranging the air holes 31c on the optical axis OA2. When the wavelength extraction filter 32 is provided, a part of the wavelength region where the spectral characteristic curves 35G and 35R cross over is measured, but this light amount is negligible and can be ignored.

  Further, as shown in FIG. 12, the camera C1 for detecting the faint light emitted from the sample 1 and the camera C2 for capturing the bright field image of the sample 1 can be switched by a slider or the like. One bright field image may be captured in color. The camera C2 is, for example, a three-plate color CCD camera having CCDs 34a to 34c, and each CCD 34a to 34c is preferably a high-definition CCD having smaller pixels than the CCD 3. The camera C2 may be a monochrome CCD camera having a high-definition monochrome CCD.

  FIG. 12 is a schematic diagram showing a partial configuration when the feeble light sample imaging apparatus according to the second embodiment includes a camera C3 for spectrophotometry together with the camera C1. As shown in FIG. 12, this weak light specimen imaging device includes a spectral cube 36 integrally having wavelength extraction filters 32 and 33 and a dichroic mirror 35 between an objective lens 6 and an imaging lens 7, and a specimen. 1 includes an imaging lens 37 for telecentric imaging of weak light emitted from 1 and reflected by a dichroic mirror 35 with a numerical aperture NAi, and a camera C3 having the same characteristics as the camera C1. Here, the dichroic mirror 35 uses a wavelength of about 600 nm corresponding to the intersection of the characteristic curves 35G and 35R shown in FIG. 11 as an inversion wavelength of transmission and reflection, transmits a wavelength longer than the inversion wavelength, and is shorter than the inversion wavelength. Reflects wavelength.

  In the weak light sample imaging apparatus shown in FIG. 12, for example, when the multicolor luciferase having the light emission characteristics shown in the characteristic curves 35G and 35R in FIG. 11 is observed as the sample 1, red light emitted from the multicolor luciferase is emitted by the camera C1. In addition to being able to measure light, the camera C3 can simultaneously measure green light emitted from multicolor luciferase. It should be noted that it is desirable to calibrate and remove differences in imaging magnification and the like due to individual differences in the characteristics of the cameras C1 and C3 and the imaging lenses 7 and 37 in advance.

  The spectral cube 36 is preferably detachably disposed on the optical axis OA2 between the objective lens 60 and the imaging lens 70. Further, together with the spectral cube 36, a plurality of replacement spectral cubes having different optical characteristics such as spectral characteristics are integrally held, and one of the spectral cubes is selectively interposed between the objective lens 60 and the imaging lens 70. A spectral cube switching device may be provided. Further, the spectroscopic cube switching device may be configured such that the dichroic mirror and the wavelength extraction filter can be individually replaced.

  FIG. 13 is a schematic diagram showing a partial configuration in the case where the feeble light sample imaging apparatus according to the second embodiment includes a camera C4 for observation of a bright field image together with the camera C1. As shown in FIG. 13, the weak light sample imaging device includes a mirror 38, an imaging lens 37, a camera C <b> 4, and an absorption filter 22. The absorption filter 22 is detachably disposed on the optical axis OA2 between the mirror 38 and the imaging lens 70 by a switching device 39 such as a slider. The mirror 38 is detachably disposed on the optical axis OA2 between the objective lens 60 and the imaging lens 70 by insertion / removal means (not shown), and when the mirror 38 is disposed on the optical axis OA2, The light is reflected toward the imaging lens 37. The specimen 1 is bright-field illuminated by the illumination fiber 15 when the mirror 38 is disposed on the optical axis OA2.

  The camera C4 is, for example, a monochrome CCD camera having a high-definition CCD with smaller pixels than the CCD 3, and takes a bright field image of the sample 1 when the mirror 38 is disposed on the optical axis OA2. On the other hand, when the mirror 38 is not disposed on the optical axis OA2, the camera C1 captures an image of the sample 1 by self-emission. At this time, the absorption filter 22 and the switching device 39 may be replaced with the filter unit 31 shown in FIG. 10, and the faint light from the sample 1 may be spectrophotometrically measured. The camera C4 may be a three-plate color CCD.

  In general, a high-sensitivity cooled CCD camera used as the CCD camera 4 has a large housing and, for example, it is difficult to replace the camera as shown in FIG. 10, but the weak light sample imaging apparatus shown in FIG. By inserting / removing on / from the optical axis OA2, it is possible to easily switch the camera to be used, and it is possible to easily switch between the bright field image of the sample 1 and the self-light-emitting image.

  As described above, in the weak light sample imaging unit and the weak light sample imaging device according to the second embodiment, the imaging optical system including the objective lens 2 (or 60) and the imaging lens 3 (or 70) is provided. Since the infinity correction system is employed, various optical elements are arranged between the objective lens 2 (or 60) and the imaging lens 3 (or 70) to make this weak light sample imaging device multifunctional. it can.

  On the other hand, the feeble light sample imaging device according to the second embodiment is arranged in a light shielding device such as a chamber that blocks light from the outside as shown in FIG. The weak light can be detected accurately and stably without being received, and an image of the specimen 1 by self-emission can be clearly captured. Here, the light shielding device shown in FIG. 14 forms a dark room by the base 41, the enclosure 42, and the lid 43, and includes a weak light sample imaging device on the base 41 in the dark room. In this shading device, the specimen 1 can be exchanged by lifting the knob 45 to open the lid 43 around the hinge portion 44.

  As shown in FIG. 14, when the weak light sample imaging device according to the second embodiment is arranged in the light shielding device, the control device 46 such as a computer having an input device 47 such as a keyboard and a mouse is used. It is preferable that the weak light sample imaging device can be remotely operated and automatically controlled from the outside of the light shielding device, and in particular, focusing and positioning operations with respect to the sample 1, imaging operations of the camera C1, etc., illumination light by the illumination fiber 15 It is desirable to be able to automatically control dimming.

  Here, the alignment with respect to the specimen 1 means that at least one of the imaging optical system including the objective lens 60 and the imaging lens 70, the camera C1, and the sample stage 13 holding the specimen 1 is substantially orthogonal to the optical axis OA2. In this process, the specimen 1 is disposed in the field of view of the objective lens 60 by moving in the direction. By using such an alignment process, the center of the Airy disk and the center of the CCD pixel are matched for each Airy disk of interest among the Airy disks formed by the imaging lens 70. May be. In this case, for example, the position where the output corresponding to the pixel is maximized may be detected by relatively operating the Airy disk and the pixel two-dimensionally.

  Further, as shown in FIG. 14, the control device 46 may include a display device 48 so that images corresponding to the bright field image of the sample 1 and the self-luminous image can be superimposed and displayed. Furthermore, the control device 46 may include a storage unit such as a memory for storing these images.

  On the other hand, in the weak light specimen imaging apparatus according to the second embodiment, instead of the sample stage 13, for example, as shown in FIG. The specimen 1 may be held on the petri dish 51. Here, this sealed container includes an air supply pipe 54 for supplying constant temperature and low humidity CO2 generated by an air conditioner (not shown) into the container, and an exhaust pipe 55 for exhausting CO2 in the container. At least one of temperature, humidity, atmospheric pressure and CO2 concentration can be adjusted. Instead of supplying and exhausting CO2, a heat sheet or the like may be provided inside the sealed container to electrically adjust the temperature in the container.

  Here, FIG. 16 systematically shows the configuration of each part suitable for application to the weak light sample imaging unit and the weak light sample imaging device according to the second embodiment. As shown in FIG. 16, the objective lens 60 has a specification of “20 times, NA 0.8, f10 mm” centering on the objective lens 601 which is a specification of “Oil, 40 times, NA 1.4, f5 mm”. It is preferable to use the objective lens 602 and the objective lens 603 having a specification of “5 times, NA 0.15, f40 mm” that can observe the entire specimen 1. As these objective lenses 601 to 603, microscope objective lenses that are generally commercially available and whose pupil positions are substantially equal to each other can be used. In addition, an objective lens 60 having a specification of “Oil, 20 ×, NA 1.4, f10 mm” that allows a large observation field of view may be used. However, in this case, the exit pupil diameter of the objective lens 60 is enlarged to 28 mm, and the diameter of the imaging lens 70 and the optical element disposed between these lenses is enlarged together with the objective lens 60, and is used in a general microscope. Optical units, optical elements, etc. cannot be used. The objective lens 6 may be provided with a variable aperture stop with a variable aperture so that the numerical aperture NAi of the imaging lens 70 can be finely adjusted.

  As shown in FIG. 16, the imaging lens 70 has a specification of “NA 0.3, f25 mm (total magnification 5 times when using a 40 × objective lens, real field of view 0.75 mm, number of fields 3.75)”. An imaging lens 701 having specifications of “NA 0.2, f35 mm (total magnification 7 times when using 40 × objective lens, real field of view 0.75 mm, field number 5.25)”, “NA 0. 1, f70 mm (image magnification 703 when using 40 × objective lens, real field of view 0.75 mm, number of fields 10.3) ”imaging lens 703,“ NA 0.15, f50 mm (40 × objective lens) It is preferable to use an imaging lens 704 or the like having a specification of “total magnification 10 times when used)”. For bright field observation, an imaging lens 705 having a specification of “NA 0.03, f100 mm (total magnification 2.5 times when using a 5 × objective lens, real field of view 6 mm, number of fields 15)” may be used. When an image is picked up using a 2/3 inch CCD as the CCD 3, if the imaging lens 7 has a field number of 10.3, slight vignetting occurs at the four corners of the image, and the field number is less than 10.3. If the image becomes round and the number of fields of view is greater than 10.3, an image without vignetting can be captured. Further, when the objective lens 6 having the specification of “Oil, 20 ×, NA 1.4, f10 mm” is used, if the lens having the specification of “NA 0.2, f70 mm” is used as the imaging lens 7, “total magnification 7 Double, 1.5 mm real field of view, 15 "field of view.

  The intermediate lens barrel disposed between the objective lens 60 and the imaging lens 70 includes an epi-fluorescent device including the fluorescent cube 24 shown in FIG. 9, the filter unit 31 shown in FIG. 10, and FIG. 12. A spectroscopic unit including the spectroscopic cube 36 and the like, and a mirror switching bright field imaging unit including the mirror 38 and the like illustrated in FIG.

  The camera that captures the image of the specimen 1 uses the cameras C1 and C3, which are high-sensitivity cooled monochrome CCD cameras, as the camera that captures the self-luminous image of the specimen 1 that is weak light. A camera C2 that is a three-plate color CCD camera or a camera C4 that is a high-definition monochrome CCD camera may be used. The size of the pixels of the cameras C1 and C3 is preferably set to about 6 to 9 μm square according to the size of the Airy disk formed by the imaging lens 7.

  As the illumination means for illuminating the specimen 1, in addition to the illumination fiber 15 that guides illumination light from a white light source or the like, as shown in FIG. 16, an illumination device that realizes critical illumination by combining a white LED and a single lens. Alternatively, a microscope UCD that is a Koehler illumination device using a condenser lens may be used. Moreover, you may provide the illuminating device which performs differential interference observation and phase difference observation. However, in this case, it is necessary to provide a dedicated ring stop, prism, objective lens, and the like.

In addition, although the weak light sample imaging unit and the weak light sample imaging device according to the second embodiment have been described as the inverted type so far, they may be the upright type. The advantage of the upright type is that it makes it easy to perform observation from above without disturbing the membrane in the observation of the tissue section using the membrane for improving the grounding to the sample container. For tissue sections, it is extremely important to be able to capture luminescent images for a long period of time while culturing for a long period of time, preferably using a living tissue having a cell layer sliced to a thickness close to one layer. I can say that. Examples of such tissue sections include central system tissues such as the cerebellum and suprachiasmatic nucleus, and various organ system tissues such as pancreas and organ tumors. Small animals or insects having light transmissivity such as nematodes can be observed without slicing. Since the present invention also provides an optical system having a wide field of view, there is a great advantage that a biological sample maintaining such exercise ability can be observed for a long period of time with a non-toxic luminescent image.

  In the weak light sample imaging unit according to the first and second embodiments described above, the CCD is used as the imaging unit. It may be an image sensor having an imaging sensitivity equivalent to that of a cooled CCD at about ° C.

  In Example 1, the condition of the square of the objective lens (NA / β) was examined so that the luminescence of the HeLa cell into which the luciferase gene was introduced could be observed with an image.

  The imaging target (Sample 1 in the above-described embodiment) in Example 1 is a HeLa cell transfected with a firefly luciferase gene “pGL3 control vector” (Promega (company name)). In observing the luminescence image of the HeLa cells, the HeLa cells were cultured for 1 day after transfection, washed with Hanks balanced salt solution, and then replaced with Hank's balanced salt solution containing 1 mM luciferin. The conditions of the numerical aperture (NA) and projection magnification (β) of the objective lens (objective lens 2 in the above-described embodiment) used in Example 1 are as shown in FIG. FIG. 17 is a diagram showing conditions for the numerical aperture (NA) and the projection magnification (β) of the objective lens used in Example 1. As shown in FIG. 17, the NA of the objective lens is a value from 0.074 to 0.4, and β of the objective lens is a value from 0.27 to 1.5. The CCD camera used in Example 1 (CCD camera 4 in the above-described embodiment) is a cooled CCD camera at 0 ° C. The number of pixels of the CCD camera is 765 × 510, the pixel size is 9 μm × 9 μm, and the chip The area is 6.89 × 4.59 (mm 2), and the quantum efficiency is 550 nm and 55%. In Example 1, the imaging of HeLa cells was performed in a state where the entire device to be used (see, for example, FIG. 1) was covered with a dark screen.

  In Example 1, as shown in FIG. 17, it was shown that the emission image can be observed under the condition of all the (NA / β) squares studied. As a result, it was shown that when an objective lens having a square of (NA / β) of 0.071 or more is used, it is possible to observe a luminescence image of a HeLa cell into which a luciferase gene is introduced.

  In this Example 2, the condition of the square of the objective lens (NA / β) so that the light emission of the HeLa cell into which the luciferase gene was introduced can be observed with an image was examined with different exposure times.

  The imaging target in the second embodiment is the same as that in the first embodiment described above. The conditions of the numerical aperture (NA) and projection magnification (β) of the objective lens used in Example 2 are as shown in FIG. FIG. 18 is a diagram showing conditions for the numerical aperture (NA) and the projection magnification (β) of the objective lens used in Example 2. Note that the objective lens used in Example 2 corresponds to “0.4X”, “0.83X”, and “1.5X” described in FIG. 17 in Example 1 described above. The CCD camera used in the second embodiment is the same as that in the first embodiment. The exposure time is 1 minute or 5 minutes as shown in FIG. Note that, as in Example 1 described above, in Example 2 as well, HeLa cell luminescence image capturing was performed with the entire apparatus to be used covered with a black curtain.

  In Example 2, as shown in FIG. 19, an image A taken with an exposure time of 5 minutes using an objective lens of “0.4X” and an exposure of 5 minutes using an objective lens of “0.83X”. Image B taken with time, Image C taken with an exposure time of 5 minutes using an objective lens of "1.5X" and Image D taken with an exposure time of 1 minute using an objective lens of "1.5X" It is possible to observe the emission image in all the images. As a result, it was shown that when an objective lens having a square of (NA / β) of 0.071 or more is used, it is possible to observe a luminescence image of a HeLa cell introduced with a luciferase gene even with an exposure time of 1 minute. .

  In this Example 3, the condition of the square of the objective lens (NA / β) that allows the image of the luminescence of the HeLa cell introduced with the luciferase gene to be observed with a fixed projection magnification (β) was examined. did.

  The imaging target in the third embodiment is the same as that in the first or second embodiment described above. Further, the condition of the numerical aperture (NA) of the object lens used in Example 3 is as shown in FIG. FIG. 20 is a diagram showing conditions for the numerical aperture (NA) of the objective lens used in Example 3. As shown in FIG. 20, the numerical aperture (NA) is 0.248 to 0.055. Note that the values of the projection magnification (β) of the objective lenses (A to F) shown in FIG. 20 are all 0.83. Therefore, the square value of (NA / β) of the objective lens varies from 0.089 to 0.004. The CCD camera used in the third embodiment is the same as that in the first or second embodiment. The exposure time is 1 minute. Note that, similarly to Example 1 and Example 2 described above, in this Example 3 as well, the imaging of HeLa cells was performed with the entire apparatus to be used covered with a dark screen.

  In Example 3, as shown in FIG. 21, an image A taken with an objective lens having NA of 0.248 (objective lens A in FIG. 20), an objective lens having NA of 0.207 (objective lens B in FIG. 20). ) And the image C taken with an objective lens having an NA of 0.164 (objective lens C in FIG. 20), the emission of HeLa cells could be easily confirmed. On the other hand, an image D taken with an objective lens having an NA of 0.121 (objective lens D in FIG. 20), an image E taken with an objective lens having an NA of 0.083 (objective lens E in FIG. 20), and NA of 0. Regarding the image F taken with the 055 objective lens (objective lens F in FIG. 20), it was difficult to confirm the light emission. As can be seen from FIG. 21, when an objective lens having a projection magnification (β) value of 0.83 is used and the exposure time is 1 minute, the square value of (NA / β) is preferably 0.01 or more. Was 0.039 or more, it was shown that luminescence image observation of HeLa cells into which the luciferase gene was introduced was possible.

  Here, from FIG. 22 which shows the relationship between the square value of (NA / β) of the objective lens used in Example 3 and the average emission intensity of the circled region of the image A shown in FIG. The condition of the square of (NA / β) of the objective lens that enables observation of the luminescent image was examined. FIG. 22 is a diagram showing the relationship between the square value of (NA / β) of the objective lens used in Example 3 and the average light emission intensity in the circled region of the image A shown in FIG. Here, in FIG. 22, the relative emission intensity is a standardization of other measured emission intensities, with the maximum value of the measured emission intensity being 1. The half value in the numerical range of the measured luminescence intensity (the numerical range of the relative luminescence intensity of about 0.6 to 1.0) is 2 (NA / β) of the objective lens that can observe the luminescence of the HeLa cell. Assuming the power condition (lower limit), the square value of (NA / β) giving the half value in Example 3 was 0.05. Thus, when an objective lens having a projection magnification (β) value of 0.83 is used and the exposure time is 1 minute, the square value of (NA / β) is 0.01 or more, preferably 0.039. As described above, it was shown that it is possible to observe a luminescence image of a HeLa cell introduced with a luciferase gene.

Industrial Applicability As described above, the luminescent sample imaging method, the luminescent cell imaging method and the objective lens according to the present invention use, for example, a luminescent gene such as luciferase as a reporter gene and a promoter or enhancer that controls gene expression. And reporter assays for examining the effects of effectors such as transcription factors and various drugs.

Next, the applicable range based on the gist of the present invention will be described.
Examples of Cells and Samples for Image Analysis The systems and methods of the present invention can be readily adapted to image any of a variety of cells provided in a variety of different ways depending on the type of substrate. For example, the cell can be a bacterial, protozoan, fungal prokaryotic cell, or eukaryotic cell, and is a bird, reptile, amphibian, plant, or mammal (e.g., primate (e.g., human), rodent It can also be a cell derived from a species (eg, mouse, rat), rabbit, ungulate (eg, cow, sheep, pig, etc.). The cells can be primary cells, normal and cancerous cell lines, genetically modified cells, and cultured cells. These cells include various cell lines that are induced spontaneously, or various cell lines that have been selected from the individual cell lines for the desired growth or response characteristics. Multiple cell lines derived from similar but different patients or sites are included. The cells are usually cultured in a sterilized environment at, for example, 37 ° C. in an incubator containing a moisturized atmosphere of 92 to 95% air / 5 to 8% CO ₂ . Culturing of the cells can be performed in a nutrient mixture containing a biological fluid such as fetal bovine serum with unspecified components or in a serum-free medium in which all components are known.

  Of particular interest is the imaging of neural cells and neural progenitor cells. The cell to be imaged can be a genetically modified cell (eg, a recombinant cell). Of particular interest is live cell imaging, although the present invention also contemplates cell membrane permeabilization or fixed cell imaging in some embodiments. The cells are usually imaged in a sample containing a culture medium intended for cell maintenance and / or growth.

  In many embodiments of the invention, particularly those embodiments that include returning to the same cell field and / or the same individual cells in the cell field, the cells are well immobilized on the substrate surface, e.g., the substrate or (e.g., ( It is attached to the treated substrate (by coating with a substance that adheres to the cells) so that the cells do not move relative to the substrate when the substrate is manipulated. For example, cells are directly attached to a substrate, eg, tissue culture plastic (eg, in a well), and the cell position is fixed relative to the substrate so that the cell position relative to the substrate does not move. Be ready to operate. In this way, it is possible to return accurately to the same cell field, or return to the same individual cell in the cell field.

  The present invention is generally intended for single cell level imaging, and in particular for live cell imaging. The cells may be dispersed as isolated single cells on the substrate surface, may be in contact with other cells (e.g., as in a monolayer), or (e.g., as in a tissue section). In addition, a thin layer may be formed. The cells to be imaged may be a homogeneous cell population or a heterogeneous cell population (eg, a mixed cell culture). Thus, the present invention allows for single cell imaging and cell population imaging, which may optionally include a plurality of different cells.

  Cell imaging may be performed with or without a detectable marker, such as a fluorescent label. Such detectable markers and methods of utilizing detectable markers with cells are well known in the art. Detectable markers include fluorophores (or fluorescence) (herein by way of example and not limitation), chemiluminescent bodies, and other suitable detectable labels, Examples include labels used in FRET (Fluorescence Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy Transfer) detection systems.

The systems and methods of the present invention can be used to image cell populations and individual cells, particularly cell viability (e.g., cell viability and cell health), cell physiological properties (synaptic physiological properties), To observe signal transduction, organelle location and function, protein location and function (including interactions and turnover), enzyme activity, receptor expression and location, cell surface changes, cell structure, differentiation, cell division, etc. It is possible to perform imaging over time. For example, in one embodiment, the systems and methods of the present invention may be used to determine whether protein expression (e.g., the role of huntingin in Huntington's disease) and protein level changes or aggregation cause cell death. Or they are used to determine if they are symptoms of cell death (eg, an attempt by a cell to prevent cell death, not the cause of cell death itself). Of particular interest is the study of neuronal degeneration of neurons in culture.

  The systems and methods of the present invention allow single cells or populations of cells to be imaged in real time at a desired time, such as a relatively short time. For example, if a 24-well substrate contains 13 optical fields in contact with each other imaged by a CCD, the wells can be imaged in about 10 minutes. In another case, the time required for cell imaging depends on the total area covered, the resolution of the CCD, and what is chosen as the focusing routine. In either case, data acquisition with the present invention is in units of seconds (e.g., only about 1 to 3 seconds per field on average in this example), focusing and moving to the next position Considering the time, the actual imaging takes less time. While it takes up to about 10-15 seconds to complete the focusing process, the time taken to capture the image of the field of view is about 50 ms to 1 second, neglecting the time required to move between fields of view. be able to. Therefore, even if another field of view is imaged, from the viewpoint of consumption time, there is substantially no problem unless refocusing is performed.

  Rapid imaging of cells, eg cells located in adjacent wells, can be performed again in a relatively short time (by returning to the same cell field containing the same individual cells) Since imaging can be performed, it has become possible to perform observations that could not be performed by the conventional method due to reasons such as the length of time required to acquire each image. In the present invention, cellular phenomena such as cell function, cell survival, and fate of individual cells in a population can also be traced over a relatively short period of time as described above. This is a result of traditional immunocytochemistry studies where only images taken at a specific time are available, and the amount of information obtained about a progressive phenomenon (e.g., denaturation) is limited, requiring time. (E.g., a study on neurodegeneration typically required about 6 weeks to analyze 300,000 cells, but using the present invention, this same analysis is much less than half) Can be completed in time). Using aspects of the present invention, when an immunocytochemical analysis or an analysis using a microscope is performed manually, an operation that normally takes 6 days can be completed in 1 hour in the processing time of a microscope and a computer. it can.

  Also, in the system and method of the present invention, a single cell and a selected cell can be selected because the substrate can be removed from the system and then placed back on the system to accurately identify the same cell population and individual cells in the cell population. Analysis over long periods of cell populations (eg, periods of hours to days to weeks or more) is also possible.

  In the systems and methods of the present invention, a plurality of biological variables (eg, parameters or variables of cell function) can also be measured (qualitatively or quantitatively) at about or exactly the same time. For example, information about changes in cell morphology and molecular phenomena can be obtained by imaging cells using both phase difference and fluorescence. In another embodiment, the cells can be imaged using multiple detectable markers (eg, multiple fluorescent markers).

  The system and method of the present invention prevents per-user bias and variation associated with conventional systems by sequentially acquiring images of the same cell or cell population. In addition, the present invention can be used for imaging cells that are sensitive to light and intense manipulation. Specifically, the exposure to a light source for obtaining an image is relatively small, and the cells It is only necessary to move the substrate on which is placed relatively finely.

Kits The present invention also provides kits for use in the present invention. Such a kit preferably includes at least a computer-readable medium including instructions and a program for realizing or implementing the above-described functions. The instructions may include instructions for installing or setting up software to program a normal microscope or cell scanner to function as described above. The instructions may also include instructions for operating the microscope as desired. The instructions preferably include both types of information.

  Providing software and instructions as a kit has several implications. This combination can be packaged and sold as a means to upgrade an existing microscope. A complete program or part thereof (preferably including at least the code defining the method of the present invention alone or in combination with code already available) can be provided as an upgrade patch. This combination can also be provided in the form that the software accompanies a new microscope pre-installed in the microscope, in which case the instructions serve as a reference manual (or part thereof) and are pre-installed It also serves as a computer-readable medium that is a backup copy of a utility.

  The instructions are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. In this case, the instructions can be provided as an attachment to the kit or as a label on the kit container or part thereof (ie, associated with the packaging or internal packaging). In another embodiment, the instruction is provided as an electronic storage data file on a suitable storage medium that can be read by a computer such as a CD-ROM or a diskette, including the same medium on which the program is mounted.

  In yet another embodiment, the instruction itself is not included in the kit, and a means for obtaining the instruction from a remote source through the Internet or the like is included in the kit. An example of this embodiment is a kit that includes a web address where instructions can be viewed and / or downloaded. Conversely, a means of obtaining the program of the present invention from a remote source can be provided, for example, by providing a web address. In addition, both instructions and software can be kits that are obtained or downloaded from remote sources such as the Internet or the World Wide Web. Of course, access security or ID protocols may be used to limit access to those who have the right to use the present invention. As in the case of the instruction sheet, the means for obtaining the instruction sheet and / or the program is usually recorded on an appropriate recording medium.

Application Examples of Imaging System and Method of the Present Invention The imaging system and method of the present invention are useful in various settings using a wide variety of cells. In the systems and methods of the present invention, cells or cell populations in tissue culture can be any desired period of time, e.g., 2 hours, 5 hours, 12 hours, 24 hours, 2 days, 4 days, 6 days, 7 days, numbers. It is also possible to follow over a period of weeks and / or to the lifetime of the cells of interest. Imaging of biological samples including cells can be performed at a certain time corresponding to the above, or can be performed in another manner . The following are examples of such imaging, but the invention is not limited thereto, and the following imaging examples highlight certain advantages and features of the invention.

Cell imaging to prevent or reduce phototoxicity Phototoxicity has always been an important limiting factor in imaging live samples, and phototoxicity is the intensity of incident light, irradiation time, wavelength. Directly related to The system and method of the present invention is used to reduce the amount of incident light required to detect available signals. Phototoxicity is a particular problem when investigating a process that progresses gradually, since the same cell sample must be repeatedly irradiated. In the present invention, phototoxicity is significantly reduced by several methods. By irradiating low intensity white light for a very short time, the microscope is focused, and then by irradiating more intense light, a high resolution fluorescent image can be obtained. Without automation, focusing will usually be achieved by continuous illumination of high intensity fluorescence. Considering the time it takes to focus the microscope and then acquire an image, the cells can be exposed to high-intensity light with phototoxicity for an order of magnitude longer than when automated. It will be.

  In addition, the method of the present invention that acquires a plurality of adjacent fluorescent images without focusing again after focusing once is also advantageous in reducing phototoxicity. This approach can be said to be an optimized method for acquiring fluorescent images because most of the cell's field of view receives only the light necessary to generate the image as significant light. Finally, since the irradiation time of high-intensity light is substantially reduced by automation, light fading is less likely to occur. Since the emitted fluorescence is bright, the excitation time required to produce a high resolution image is further reduced.

Optical imaging of gene expression by stimulation The present invention can be carried out, for example, as follows.
A reporter gene (preferably a firefly or a reporter gene) operably linked to a cell constant (for example, a promoter region of a gene whose expression is induced by stimulation with which the substance is brought into contact with the cell (preferably a promoter region of the c-fos gene) A luciferase derived from Renilla, etc. is introduced into a cell using the above-described gene introduction method, and constants (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ^{3 to} 1 × 10 ⁶ cells) of the obtained cells into which the gene has been introduced are obtained. ) Is cultured in a desired nutrient medium (eg, D-MEM medium) using an apparatus capable of culturing the desired cell (eg, petri dish, multi-plate having a large number of wells, etc.) In advance, keep the sample at a temperature optimal for the cells (for example, 25 to 37 ° C, preferably 35 to 37 ° C), and inject water to prevent the sample from drying. A luminescence image is recorded by a digital camera through an objective lens in a sample observation section of the luminescence microscope, and a substance for stimulating the sample by contacting the cell (for example, , Compound) at a desired concentration (eg, 1 pM to 1 M, preferably 100 nM to 1 mM), and recording a luminescent image at a desired time interval (eg, 5 minutes to 5 hours, preferably 10 minutes to 1 hour). The recorded image is acquired using a commercially available image analysis software (for example, MetaMorph; manufactured by Universal Imaging Co., Ltd.) to obtain the luminance value in a desired area in the image, and also the bright field image is captured in the same field of view as the luminescent image. If the image analysis software has a function to superimpose the light emission image and the bright field image , Recognize cells by using clear images of bright-field images even for unclear luminescent images of fast-moving cells (whole tissue, specific cells, individual cells, partial areas of cells, etc.) In this way, the image analysis software for performing image analysis using the imaging method and apparatus according to the present invention has individual advantages in at least a luminescent image. A recognition function that identifies cells (whole tissue, specific cell groups, individual cells, partial areas of cells, etc.) by contour information based on parameters such as shape and size, and luminescence generated from recognized cells A measurement function for measuring the amount, preferably from a computer that controls the imaging device or an input means (keyboard, mouse, numeric keypad, touch panel, etc.) by the operator. And outputting the measurement result according to. More preferably, the measurement result is output in association with the image information of the recognized cell or the like. The output format may be a pseudo image or a numerical value corresponding to the amount of luminescence, and in the case of analyzing a large number of cells, it may be a graphic expression such as a normal distribution, a histogram, a line graph, or a bar graph. Further, when outputting time-series analysis results regarding the same cells or the like, a point distribution in which light emission amounts are arranged in order of elapsed time or a waveform pattern connected by lines in time order may be used. The waveform pattern is particularly suitable for light emission data exhibiting periodicity such as a clock gene. If necessary, the image analysis software may be configured to analyze the correlation with other cells or the like, independently of the output graph or waveform pattern. Furthermore, it is preferable to have a recall function for displaying on the display an image of a cell or the like corresponding to a portion of interest in the analysis result output on the display when it is designated through the input means. It is further preferable that the recall function has a screening mode in which moving image information is displayed for the entire period or a specific period in which the designated cells are imaged.

Long live imaging of living cells Live cells are often cultured on tissue culture plastic. When examining live cells, especially when examining live cells in a progressive process, it is necessary to maintain the health of the live cells (eg, nerve cells) for a period of time sufficient to cover the process being examined. is there. Ideally, imaging cells over a period of time in the same culture dish where the cells were originally grown will maintain cell health and minimize disruption. It is possible to image cells in tissue culture dishes using an inverted microscope under sterile conditions, but in the case of an inverted microscope, image through the substrate on which the cells are grown (e.g. glass or plastic). Will do. Compared to glass, tissue culture plastics are inferior in transparency (for example, ultraviolet rays) depending on the wavelength, cause light scattering, and reduce image resolution. However, many cells, including nerve cells, are longer when grown on tissue culture plastic than glass, even when the substrate is coated with polylysine and laminin to promote cell attachment and differentiation. Survive over and show a healthy appearance. Accordingly, an object of the present invention is to provide a system including an optical system that can obtain a high-quality image regardless of whether plastic or glass is transmitted.

When images are automatically acquired through glass or plastic, there is a significant impact on the objectives that can be used. In other words, an immersion lens usually collects more light than a non-immersion (air) objective lens, but an immersion lens requires an immersion solvent, and in the case of automatic imaging, supplying this solvent is a reality. Not right. The task of focusing through substrates having various compositions and thicknesses using non-immersion lenses is also problematic. The refractive index of these substrates differs from product to product, and is different from the refractive index of air that passes until the emitted fluorescence is collected on the objective lens. Imaging through materials of different refractive indices results in chromatic and spherical aberration, which increases the numerical aperture of the lens. Aberrations become apparent at 20 times, become substantial at 40 times, and become almost impossible to overcome at 60 times. Finally, some specimens, such as cultured brain slices, are located relatively far from the bottom of the tissue culture dish and can be viewed from various planes along the Z axis using an algorithm that automatically determines the focal plane. Need to get an image. In such a case, it is preferable to apply the optical convolution function to the light emission imaging data of the present invention. By adopting the optical convolution function, a three-dimensional image can be provided not only for a section but also for a living tissue or a small living organism (insect, animal, plant, etc.). Note that when an objective lens having an extremely long focal length is used, it is possible to focus on a distant object, and it is possible to prevent the objective lens and the tissue culture plate from colliding during automatic focusing.

High throughput screening assays The systems and methods of the present invention are particularly useful in performing high throughput screening assays. Examples of such assays include substances that elicit the desired cellular response (eg, regulation of cell death, receptor internalization, regulation of signal transduction pathway activity (increase or decrease), regulation of transcriptional activity, etc.) And analysis of nucleic acids with previously unknown or unexamined functions (e.g., analysis in which a coding sequence of interest is introduced into a target cell and expressed in the cell) However, it is not limited to these. A cell in which a response is observed is referred to herein as a “target cell”, but this is not a term for limiting the cell type, but rather suggests that the substance is affected by the substance. Is the term.

In general, using the systems and methods of the present invention, the effects of substances in living cells can be analyzed for the same cells at desired time intervals. Here, the cells can be a homogeneous cell population or a mixed cell population. The assay method of the present invention has an effect that a cell modified by a substance gives to other cells in culture, for example, the influence that expression of a nucleic acid substance in a target cell has on a target cell or other cells in the same well. You can also find out about. For example, the assay of the present invention can be used in detecting a “bystander effect” in which cells modified by a substance have an effect on cells that are not directly modified by the substance, Here, the latter cells may or may not be in physical contact with the cells modified by the substance. In this context, the assay methods of the invention can be used, for example, in examining secreted proteins or cell surface proteins induced by a code or substance.

Detection of multiple variables in a screening assay The imaging system and method of the present invention can be used to obtain data for multiple variables in a single sample, for example, one or more biological variables Can also be detected, and changes in such biological variables detected over time, for example, can also be detected by comparing images obtained by using the system of the present invention.

  By way of example, the system of the present invention may be caused by changes in biological material over time, such as changes caused by the contact of a material with a particular substance (e.g., increased concentration of one substance, addition of another substance, etc. Change) and changes caused by changes in environmental conditions (for example, adjustment of temperature, osmotic pressure, etc.) can be used. The change in biological material can be, for example, for nucleic acids, the accessibility of a particular nucleic acid sequence by a detectable probe, the degree of supercoil or duplex, etc. For proteins, the change can be the degree of protein folding, access to the binding site of the probe (eg, a detectable antibody or other protein binding agent), and the like.

  For example, for a population of cells or variables related to cells in a single cell (e.g., one or more variables for a cell, two or more variables, etc.), a screening assay can be performed for multiple cell variables of a single cell. These cellular variables can be examined at various times as needed. In general, cellular variables can be detected, particularly biologically active, cellular components, or cells that can be detected with sufficient accuracy, preferably in a manner suitable for the high-throughput assay of the present invention. It can be a product. Examples of cell variables include cell health (eg, life-and-death distinction detected by permeability of cell membranes to dyes such as trypan blue or ethidium bromide, induction of apoptosis, etc.); status of cell surface receptors ( For example, receptor binding, activation, reuse, etc .; gene transcription level (eg, level detected with a reporter gene (eg, GFP fusion protein)); cell differentiation (eg, formation of cell structure (eg, neuronal cell) Dendrites formation), status detected by the presence or absence of cell differentiation antigen); transfection status (i.e. presence or absence of recombinant polynucleotide used for analysis in target cells), etc. There is. References to “cellular variables” herein are not intended to be limiting in any way, but are only for convenience of explanation and clarity of content.

  Most of the variables provide quantitative reading results, but semi-quantitative or qualitative results are sometimes acceptable. The read result may include a single determination result, or may include an average, median, or variance. A range of parameter readings for each parameter is characteristically obtained by performing the same assay combination multiple times, usually by obtaining at least about twice the same assay combination. Values are expected to vary, and the range of values for each set of test variables uses standard statistical methods together with common statistical methods used in obtaining a single value It is obtained by doing.

  Whether the cellular parameter to be analyzed generally modulates the physiological condition being simulated by combining the following criteria: assay; factors available and known to regulate the parameter in vitro Whether the parameter is regulated in vivo in a manner similar to that regulated (eg, as a control); is assured as detectable and distinguishable from other analyte cell variables It is selected depending on whether there is such a response; and in some cases, whether the change in parameters is indicative of toxicity that leads to cell death, especially in drug screening assays. Note that the cell parameters do not necessarily have to meet all these criteria.

  When evaluating multiple parameters, different variables can be detected using markers that can be distinguished during detection. For example, if the screening assay involves an assessment of the effect of the gene product encoded by the polynucleotide, one marker is used to identify cells that have been transfected with the construct of interest (eg, the polynucleotide of interest Of a transfected cell in a construct of interest by use of a detectable marker encoded in the construct comprising or use of a detectable marker present on a construct co-transfected with the construct of interest Identification) and using a second marker to detect expression of the gene product (e.g., detection of the gene product in a detectable marker provided by a fusion protein generated from the gene product encoded by the polynucleotide) Target gene products using a third detectable marker The effect on the cell can be assessed (e.g., when assessing cell viability, expression of a reporter gene under the control of a promoter that is assumed to be regulated by the gene product of the candidate polynucleotide) Or by factors that are regulated by the gene product of the candidate polynucleotide). It is also possible to obtain information about changes in cell morphology (e.g., cell differentiation and cell structure (e.g., dendrite) formation) from a phase contrast image. And can be compared with the fluorescence image acquired at the selected time.

  In another example of identifying a substance that modulates the activity of a receptor on a target cell by a screening assay, a first marker is used to detect binding of the substance to the receptor, while a second marker is used. Activation by transcription of the reporter gene can be detected. As used herein, “detectable marker” includes molecules that emit a detectable signal when excited at a predetermined wavelength. In some embodiments, the same molecule may serve as multiple different markers (e.g., cells may excite and / or emit at different wavelengths when the molecules are located in different compartments of the cell). When present on the surface, the molecule emits light at a wavelength different from that of the acidic compartment in the cell).

  Various methods can be used to quantitatively examine the presence or absence of the selected marker. Fluorescent moieties that can label almost all types of biomolecules, structures, and cells are readily available. Immunofluorescent moieties can be linked not only to specific proteins, but also to specific conformations, cleavage products, specific site modifications such as phosphorylation. Individual peptides and proteins can be engineered to emit autofluorescence, for example, by expressing them as chimeras of green fluorescent protein in cells (for review see Jones et al. (1999) Trends Biotechnol 17 (12) : 477-81)).

  Fluorescence technology has now matured to the point where many useful dyes are commercially available. Fluorescent dyes are available from a number of suppliers, such as Sigma Chemical Company (St. Louis, MO), and Molecular Probes ("Handbook of Fluorescent Probes and Research Chemicals", 7th Edition, Molecular Probes, Eugene OR) Can be purchased from Other fluorescent sensors are designed to report on biological activity and environmental changes, such as pH, calcium concentration, potential, proximity to other probes, and the like. Important methods include calcium flow, nucleotide incorporation, quantitative PAGE (proteomics) and the like.

Multiple fluorescent labels may be used in the same assay, allowing cells to be detected qualitatively or quantitatively individually to allow simultaneous detection and / or measurement of multiple cellular responses. Numerous quantitative techniques have been developed to take advantage of the unique properties of fluorescence, such as direct fluorescence measurement, fluorescence resonance energy transfer (FRET), fluorescence polarization or anisotropy (FP), Examples include time-resolved fluorescence (TRF), fluorescence lifetime measurement (FLM), fluorescence correlation spectroscopy (FCS), and fluorescence fading recovery (FPR) (`` Handbook of Fluorescent Probes and Research Chemicals '', 7th edition, Molecular Probes, Eugene OR). Of particular interest are those that can be applied to live cells and in some cases can be used at a desired time (e.g., between images taken at intervals of hours or days). It is a labeling technology that can be compared. Appropriate steps or apparatus changes (or improvements) can be made so that these fluorescence-based assay methods can be applied to assay methods using luminescence that do not require photoexcitation for the present invention.

Candidate substance
The term “substance” when referring to this “candidate substance” refers to any target molecule that can be contacted with a living cell to evaluate the effect on the living cell. Since the present invention has high throughput performance, multiple evaluation mixtures of different substance concentrations are processed in parallel in different wells (e.g., in different wells of a multi-well plate) for observation. The concentration dependence of the effect produced can be examined. Usually, one of these concentrations will be a negative control, i.e. a zero concentration or a concentration below the detection concentration.

  As used herein, a candidate substance can be a number of chemical taxa, such as nucleic acids (e.g., DNA, RNA, antisense polynucleotides, etc.), polypeptides (e.g., proteins, peptides, etc.), organic molecules (e.g., Small organic compounds having a molecular weight of greater than 50 daltons and less than about 2,500 daltons), ribozymes and the like, but are not limited thereto. Candidate substances can contain functional groups necessary for structural interactions with proteins, especially hydrogen bonds, usually at least one amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two chemical functional groups Can be included. Candidate substances often have a cyclic carbon or heterocyclic structure and / or an aromatic or polyaromatic structure substituted with one or more of the above functional groups. As described above, candidate substances can be, but are not limited to, biomolecules such as polynucleotides, peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives thereof, structural analogs, or combinations thereof. Is not to be done.

  Candidate substances can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means can be used, including the expression of randomized oligonucleotides and oligopeptides, to perform random and directed synthesis of a wide variety of organic compounds and biomolecules. Alternatively, libraries of natural compounds such as bacterial, fungal, plant, and animal extracts are available or can be readily generated. Furthermore, natural or synthetic libraries and compounds can be readily modified by conventional chemical, physical, and biochemical means and used to generate combinatorial libraries. Known pharmacological substances can also be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidation to produce structural analogs.

When the candidate polynucleotide is a polynucleotide, the molecule can be in the form of a polymer of any length and can be a ribonucleotide or a deoxynucleotide. Thus, a “polynucleotide” is a single-stranded, double-stranded, or multiple-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or purine and pyrimidine base, or other natural This includes, but is not limited to, a polymer containing a chemically or biochemically modified, non-natural or modified nucleotide base. If the polynucleotide encodes a gene product, the polynucleotide may also include intron and exon sequences.

The backbone of the polynucleotide can contain sugar and phosphate groups (as commonly found in RNA or DNA), or can contain modified or substituted sugars or phosphate groups. The backbone of the polynucleotide can also include synthetic subunits, such as polymers of phosphoamidites, and can therefore be oligodeoxynucleoside phosphoramidites, or phosphoamidite-phosphodiester oligomer mixtures. For example, antisense oligonucleotides are polynucleotides that are chemically modified native phosphodiester structures in order to increase intracellular stability and binding affinity. Useful skeletal chemistry changes include phosphorothioates, phosphorodithioates in which both non-bridging oxygens are replaced with sulfur, phosphoramidites, alkylphosphotriesters, and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH ₂ -5'-O-phosphonate, 3'-NH -5'-O-phosphoramidate. In the case of peptide nucleic acids, the entire ribose phosphodiester backbone is replaced with peptide bonds. Modifications with sugars have also been used to increase stability and affinity (eg, morpholino oligonucleotide analogs). It is also possible to use a deoxyribose β-anomer whose base is inverted with respect to the natural α-anomer. The 2'-OH of the ribose sugar can be changed to become a 2'-O-methyl or 2'-O-alkyl sugar to impart degradation resistance without sacrificing affinity.

  A polynucleotide may also comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars, and linking groups, such as fluorobiose and thioate, nucleotide branches. Non-nucleotide components may intervene between the base sequences. A polynucleotide can also be modified or further modified after polymerization, such as by conjugation with a labeling component, to facilitate detection of the molecule. Still other modifications include caps, substitution of one or more naturally occurring nucleotides with analogs, and attachment of the polynucleotide to a protein, metal ion, labeling component, other polynucleotide, or substrate (e.g., a bead). Although there is introduction of means, it is not limited to these.

  In one embodiment described in detail below, a polynucleotide is screened with the assay of the present invention and the effect of the gene product encoded by the polynucleotide is assessed. In this embodiment, the polynucleotide can be provided as part of a construct suitable for expression. In this embodiment, by modifying the polynucleotide, a promoter element can be operably linked to the open reading frame of the polynucleotide encoding the gene product to facilitate expression of the gene product in the target cell.

Depending on the polypeptide embodiment as a candidate substance, the candidate substance is a “polypeptide” or “protein”. These terms are used interchangeably and refer to polymer form amino acids of any length. Examples include genetically encoded amino acids and non-genetically encoded amino acids, chemically or biochemically modified (e.g., post-translationally modified, e.g., glycosylated), or modified amino acids, Examples include polymeric polypeptides, as well as polypeptides with modified peptide backbones. “Polypeptides” that can be screened as candidate substances include effusion proteins with heterologous amino acid sequences, fused with heterologous and homologous leader sequences, with or without an N-terminal methionine residue. And non-immunologically labeled proteins. Polypeptide modifications can also be made, for example, to promote attachment to a substrate (eg, beads). If the polypeptide is not taken up into the cell, the polypeptide can be introduced into the target cell, for example by microinjection. However, such methods may be undesirable because microinjection procedures are not suitable for use in high throughput screening assays.

Cells for use in screening assays Suitable cells for use in the screening assays of the present invention include those described above. In some embodiments, assays for recombinant cells expressing a target gene product and assay methods are targeted, for example, by binding to the target gene product, modulating the expression of the target gene product, or modulating the biological activity of the target gene product. Of particular interest is making it suitable for the detection of candidate substances that interact with gene products. As used herein, “target gene product” refers to various gene products including proteins that are the center of screening for candidate substances, but is not limited thereto. For example, the target gene product can be a receptor and the assay can be adapted to identify substances that modulate the activity of the receptor.

Regardless of the purpose of the screening assay method, the general assay method In the assay process, the substance is brought into contact with the cell. Introduce into the cell and detect one or more variables of the cell. Changes in cellular parameter readings corresponding to the substance are measured, normalized if possible, and evaluated by comparing this parameter with a matching reading. Reading results for verification, basic readings obtained in the presence and absence of various factors, obtained by using other substances (which may or may not contain known inhibitors of known pathways) Readings, etc. can be used. Analytes can include any biologically active molecule that has the ability to directly or indirectly modulate the cellular parameters of interest of the cells of interest.

  The substance is conveniently added in the form of a solution to the cells or in a readily soluble form to the culture medium of the cells being cultured. The substance can be added to the flow-through system in an intermittent or continuous stream, or the compound can be added to the static solution at once or in batches, except during the addition. One example of a flow-through system uses two fluids, one with a physiologically neutral solution and the other with the test compound added in the same solution. A first fluid is passed over the cell followed by a second fluid. In a method that uses only one solution, the test compound is added to a specific volume of culture medium surrounding the cells. The overall concentration of the components of the culture medium should not vary significantly between the addition of the test compound or between the two solutions in the flow-through method.

  In some embodiments, the composition of the material does not contain additional components such as preservatives that can significantly affect the overall composition. In this case, the composition of the substance consists essentially of the substance to be tested and a physiologically acceptable carrier, such as water, cell culture medium and the like. In other embodiments, another substance can be included in the screening assay, such as a substance that allows static binding of the substance to its binding partner, non-specific mutual Examples include substances for reducing the action or background interaction. Of course, it is necessary to select such substances that are compatible with the screening of living cells.

  As described above, multiple assays using different substance concentrations can be performed in parallel to obtain differential responses corresponding to various concentrations. As known in the art, a range of concentrations obtained by dilution with 1:10 or other logarithmic scale is usually used in determining the effective concentration of a substance. This concentration can be further refined if necessary by performing a second series of dilutions. Typically, one of these concentrations serves as a negative control, which has a zero concentration, or a concentration that is below the substance detection level or below the substance concentration at which no phenotypic change can be detected. To do.

  Examples of assay methods utilizing various aspects and features of the system and method of the present invention are shown below, but the present invention is not limited thereto.

Drug Screening Assays The imaging systems and methods of the present invention are adapted for a wide variety of assay formats and screened for the desired biological effect of a candidate substance on target cells (eg, modulation of target cell parameters). This biological effect can be meaningful when the substance is used as a medicament. For example, various substances can be used for cell differentiation, cell death (for example, regulation of apoptosis), signal transduction (for example, signal transduction at the G-binding protein receptor, GTP binding, detection of the second messenger system, etc.), ion channel activity (E.g., assessing influx using, e.g., calcium imaging), transcription (e.g., using a reporter gene assay to identify substances that affect expression of the target gene product, etc.), etc. You can find out about regulation. Of particular interest are assays that can be used with living cells.

  In one embodiment, the screening assay is a binding assay that detects binding of a candidate substance to a binding partner in a cell, eg, a screening to identify substances that act as agonists or antagonist ligands to a receptor. Can do. In certain embodiments, the assay can also be an antagonistic binding assay that evaluates candidate substances for inhibition of the activity of, for example, a known receptor ligand (eg, a known agonist or antagonist). In this latter embodiment, the known ligand is labeled so that it can be detected, and the detectable label of the known ligand is reduced, for example, as the activity of the known ligand decreases or binds. it can.

  When incubating the candidate substance with the target cells, the culture can be performed at any appropriate temperature, and usually at 4 to 40 ° C. The incubation time is selected to optimize the activity, but it is also possible to select the optimal time for proceeding with rapid and high-throughput screening. An incubation time of 0.1 to 1 hour is usually sufficient, but in some embodiments it may be desirable to examine the cells after an appropriate period of time from this incubation time.

Functional Genetic Analysis In one embodiment, the imaging systems and methods of the present invention are used to implement a high-throughput functional genetic screening assay. Such assays generally involve examining genetically modified cells (for example, by stable or transient introduction of recombinant genes, or antisense technology), and the genetic modification results in biological expression in the target cell. Evaluate whether gain or loss of function has occurred. Such assays not only identify substances that may be useful as drugs (e.g., in gene therapy or antisense therapy), but also identify, for example, target genes using gain or loss of function and unknown function. It is also useful for the analysis of genes.

  Methods for producing genetically modified cells are known in the art. See, for example, Ausubel et al., “Current Protocols in Molecular Biology” John Wiley & Sons, New York, N.Y., 2000. In functional genetic analysis, in some embodiments, the candidate substance is a polynucleotide that encodes a gene product of interest (e.g., a peptide, protein, or antisense RNA) and may act as an antisense molecule, etc. It can also play a role in various drug screening assays. Specific examples of “genetic material” will be described later. This genetic modification can result in a knockout that results in a deletion that reduces the expression of the target gene as a result of homologous recombination (eg, to an undetectable level), or stably introduces genetic sequences that are not normally present in the cell. You can also knock in.

  In the present invention, a knockout body can be prepared using various methods including the expression of site-specific recombination, antisense, or dominant negative mutants. In the case of gene targeting, the knockout body partially or completely loses the function of one or both of the alleles of the endogenous gene. The expression of the target gene product is preferably undetectable or negligible in the cell to be analyzed, for this purpose the introduction of a break into the coding sequence, for example the insertion of one or more stop codons, the DNA fragment What is necessary is just to perform deletion of a coding sequence, substitution to the stop codon of a coding sequence, etc. by insertion. In some cases, the introduced sequence is eventually removed from the genome, leaving the native sequence unchanged.

Modification of cells for functional genetic analysis In general, functional genetic analysis involves the manipulation of cells by introducing nucleic acids (e.g., by expression of recombinant proteins and inhibition of expression mediated by antisense), etc. Screening for the effect of adding or losing the function of. Such a substance is referred to as “genetic material” for convenience in the present invention, but this “genetic material” is not particularly limited. The introduction of genetic material usually changes the overall nucleic acid composition of the cell. By using genetic material such as DNA, changes in the cell genome can be experimentally produced, usually by integrating the sequence into the chromosome. If the foreign sequence is not integrated and retained as episomal material, the genetic change may be transient. Genetic material, such as antisense oligonucleotides, can affect protein expression without altering cellular genotype by interfering with transcription or translation of mRNA. In general, the effect of genetic material is that it increases or decreases the expression of one or more gene products in a cell.

  The introduction of an expression vector encoding the polypeptide causes the encoded product to be expressed in cells lacking that sequence, or the product is overexpressed (e.g., expressed at a higher level than with endogenous genes alone). Can be used for. A variety of constitutive or inducible promoters can be used. These coding sequences can include full-length cDNA or genomic clones, fragments derived therefrom, or chimeras that combine native sequences with functional or structural domains of other coding sequences. Alternatively, the genetic material to be introduced should be one that encodes an antisense sequence, an antisense oligonucleotide, a dominant negative mutation, one that encodes a dominant or constitutively active mutation of a natural sequence, a modified regulatory sequence, etc. Can do.

  In addition to genetic material having a sequence derived from the host cell species, other genetic material of interest is, for example, the coding region of genetic material having a sequence obtained from a pathogen, such as a virus, bacteria, or protozoan gene. In particular, a part in which a gene affects the function of a cell of a host such as a human can be mentioned. A sequence derived from another species may be introduced when the corresponding homologous sequence is present or absent.

  Numerous public resources are available as a source of gene sequences, eg, human, other mammalian, human pathogen sequences. A substantial portion of the human genome has been sequenced and can be accessed through public databases such as Genbank. Resources can also utilize a set of unigenes and genomic sequences. See, for example, Dunham et al. (1999) Nature: 402 489-495, or Delukas et al. (1998) Science 282: 744-746. CDNA clones corresponding to many human gene sequences are available at the IMAGE consortium. An international IMAGE Consortium laboratories has developed and cloned cDNA clones for global use. Such clones are commercially available from, for example, Genome Systems Inc., St. Louis, Missouri. Methods for cloning sequences by performing PCR based on DNA sequence information are also known in the art.

  In one embodiment, the genetic material is an antisense sequence that acts to reduce the expression of complementary sequences. Antisense nucleic acids are designed to specifically bind to RNA to form RNA-DNA or RNA-RNA hybrids and inhibit DNA replication, reverse transcription, or translation by messenger RNA. Antisense molecules inhibit gene expression by various mechanisms including, for example, reducing the amount of mRNA available for translation, activation of RNAse H, or steric hindrance. Antisense nucleic acids based on the selected nucleic acid sequence can interfere with the expression of the corresponding gene. Antisense nucleic acids can be generated in cells by transcription from an antisense construct that includes the antisense strand as the transcription strand.

  The antisense substance can be an antisense oligonucleotide (ODN), in particular a synthetic ODN having a chemical modification derived from a native nucleic acid, or a nucleic acid construct that expresses an antisense molecule such as RNA. Antisense molecules can be administered alone or in combination, and when administered in combination, the administered antisense molecules can comprise multiple different sequences.

  In general, a specific region or regions of the endogenous sense strand mRNA sequence is selected for complementary pairing with an antisense sequence. Empirical techniques can be used in selecting a particular sequence for an oligonucleotide, in which case several candidate sequences are examined for inhibition of target gene expression. Combinations of sequences can also be used, in which case several regions of the mRNA sequence are selected for complementary pairing by antisense.

  Dominant negative mutations can be screened for loss of cellular function to facilitate analysis of protein function, for example. These include several different mechanisms such as substrate binding domain mutations, catalytic domain mutations, protein binding domain mutations (e.g. multimer formation, effectors, or protein binding domain activation), cell localization domain mutations It may be affected by mutations. For the creation of dominant negative mutations, general strategies can be used (see, eg, Herskowitz (1987) Nature 329: 219, and references cited above).

  Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals to increase the expression of foreign genes introduced into the cell. Such methods can include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Moreover, RNA that can encode the sequence of the gene product can also be chemically synthesized, for example, using a synthesis system. See, for example, the technique described in “Oligonucleotide Synthesis”, 1984, Gait, M. J., IRL Press, Oxford.

  Various host-expression vector systems can be utilized to express the gene coding sequence. Expression constructs include promoters derived from the genome of mammalian cells (eg, metallothionein promoter, elongation factor promoter, actin promoter, etc.), promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 late) Promoter, cytomegalovirus, etc.). In mammalian host cells, a number of viral-based expression systems can be used, such as expression systems based on retroviruses, lentiviruses, adenoviruses, herpesviruses, and the like.

  If the entire gene, ie the entire gene including the start codon of this gene and adjacent sequences, is inserted into a suitable expression vector, no additional translational control signals are required for expression in the target cell. However, if only a part of the gene coding sequence is inserted, it may be necessary to use an exogenous translational control signal, possibly including the ATG start codon. In addition, the start codon must match the reading frame of the desired coding sequence to ensure that the entire insert is translated. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcription enhancer factors, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153: 516-544).

  Using techniques that can perform transfection with high efficiency (eg, about 80-100% of the cells) avoids the need to identify transfected cells using selectable markers. Such techniques include infection with adenovirus (e.g., Wrighton, 1996, J. Exp. Med. 183: 1013; Soares, J. Immunol., 1998, 161: 4572; Spiecker, 2000, J. Immunol 164: 3316; And Weber, 1999, Blood 93: 3685), and infection with lentiviruses (eg, International Patent Application No. 000600 or 9851810). Other important vectors include lentiviral vectors. See, for example, Barry et al. (2000) Hum Gene Ther 11 (2): 323-32; and Wang et al. (2000) Gene Ther 7 (3): 196-200.

  In some embodiments, for example, when trying to examine the effects of transfected cells on non-transfected cells, or when it is difficult to accurately image a single cell due to signals detected from multiple transfected cells In some cases, it may be desirable to have low transfection efficiency. The low transfection efficiency may be less than about 25%, 20%, 10%, or 5%, and in some cases less than about 1% of the transfected cells. Since the imaging system and method of the present invention can be used for rapid identification and re-identification of the same cells, transfection efficiency is not critical in assessing the effect of genetic material on target cells.

  The following modifications are given for the complete disclosure and description of how to use the present invention to those skilled in the art, and the scope of what the present inventors regard as the invention is limited by these examples. And these examples are not all of the experiments performed below, nor do they indicate that only the following experiments were performed. The numbers used (eg, length, quantity, temperature, etc.) have been made as accurate as possible, but this does not exclude the possibility of some experimental errors and bias being included. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Modification 1: Modification concerning the numerical aperture of the objective lens
A modification in which an objective lens having a low numerical aperture is used together will be described. Lowering the numerical aperture increased the depth of field of view, and objects with a wider range in the Z-axis direction remained in focus in the image. For example, in the case of an objective lens with a low magnification and a low numerical aperture (for example, an objective lens with a magnification of 4 × and a numerical aperture of 0.13), it is possible to focus further by focusing once in the center of the well of a 24-well plate. Even without correction, the adjacent field of view of the microscope can be clearly imaged. This setting is convenient when high-throughput cell counting is performed because many fields are included in each field of view at a low magnification . In addition, this setting has an advantage that it is possible to acquire an image of another field of view in the same well without spending time by refocusing .

  For objective lenses with medium to high numerical aperture, another solution has been developed. Increasing the numerical aperture (e.g., 10x objective lens with 0.30 numerical aperture, 20x objective lens with 0.45 numerical aperture, 40x objective lens with 0.60 numerical aperture) reduces the depth of field, In other words, the difference in the position in the z-direction of cells located in the center of the well, for example, neurons, from the peripheral edge of the well is resolved, and the focal point is originally set at the center of the well. (Assuming they were), only the nerve cells located in the center of the well are clearly focused.

  We have also found that the z-direction position of the cells in the well is mainly determined by the overall tilt of the multiwell plate in the plate holder. This tilt changes slightly and unpredictably each time the plate is placed in the holder, but the plate position remains nearly constant for each imaging session where the plate stays in the holder. Met. So, using autofocus, measure the tilt of the plate in the holder based on empirical rules, and then use this measurement to determine the exact position of the well, i.e. in the xy direction in the well A program that automatically adjusts the focus position (z direction) in detail has been developed. With this program, it is possible to measure the detailed position of the focal plane in the z direction for four different x-y positions located in a single well or in the middle of four different wells. Using exact x, y, z values for four different focal positions, it is possible to calculate the focal plane change (z direction) that occurs with changes in the x or y direction. These values are incorporated into the autofocus program and the focal plane is determined empirically for the center position of each well, and the program automatically determines the focus position for all other fields in the same well. Can be calculated automatically. This method allows a high numerical aperture lens to be used with an automatic acquisition program, which is much faster than the time required to accurately focus the entire well and refocus each microscope field of view. Images can now be acquired.

Focusing on different wavelengths In the process of trying to collect images of the same field of view of the microscope using different wavelengths of fluorescence, the lens refracts different wavelengths of light in different amounts, so If the wavelengths are different, the position of the image formed by the light is not exactly the same position. Since this difference is small, it was not detected in the case of an objective lens having a low numerical aperture (for example, an objective lens having a magnification of 4 and a numerical aperture of 0.13). However, as the numerical aperture increases, these differences are resolved. Therefore, in the method or apparatus using fluorescence, when the excitation emission filter is replaced, it is necessary to rearrange the objective lens to clearly focus the field of view of the same microscope. However, in the case of the present invention, there is no need to irradiate corresponding excitation light of different wavelengths in order to detect light emission of different wavelengths, so that it is substantially unnecessary to adjust the focus for each wavelength.

The focal plane determined by automatic focusing using transmitted light can also be used as a reference when determining the position of the luminescent image . In other words, the focal plane obtained by the transmitted light may be positioned at a relatively fixed distance . At this time, the position of the focal plane with respect to the reference focal plane is determined based on an empirical rule for various emission components , and then the computer replaces the optical filter for detecting the emission and automatically introduces appropriate compensation. Such a program can be executed.

Variant 2: When the acquisition and acquisition program for accurate and high-throughput images is executed twice for the same plate without removing the plate from the plate holder, the two images obtained have almost the same field of view of the microscope. It was a thing. However, when the plate was removed once and placed between the imaging steps, the contents of the microscopic field of the image only partially overlapped. In the former case, the same microscopic field is obtained, but in the latter case, the stage movement is extremely precise, whereas the position of the plate in the holder changes. It was suggested that it was easy. Attempts to fix the position of the plate within the plate holder were not practical, and were basically ineffective.

  Rather, this problem was solved by developing a simple program that associates the operation of the image acquisition program with the internal verification points of the plate itself. For example, some multi-well plates are stamped with letters and numeric symbols adjacent to each well below the plate, depending on the manufacturer. One such stamp was used as a reference mark in our program. In this program, the stage is moved so that the position of the reference mark is imaged, automatic focusing is performed, and then a collation image of the mark is acquired. Associate all other points of the acquisition program with this reference mark. When the plate returns to the plate holder in preparation for the next imaging step, the plate is repositioned, the reference mark position is returned to the previous acquisition step, and then the acquisition program is executed.

Variant 3: Monitoring of the survival state of individual neurons As a result of being able to accurately return to the same visual field of the microscope at regular intervals at regular intervals and monitoring the survival state of individual neurons, data The degree of freedom of analysis is increasing. For example, using a low magnification objective lens (this analysis is referred to herein as a “population-based” analysis because of the large number of cells in the field of view, ie the image), or using high magnification (this In analysis, the field of view, i.e., the number of cells in the image, is referred to herein as a “single cell-based” analysis). Kaplan-Meier analysis is a well-established technique for analyzing the data obtained. Can be applied to quantify the survival promoting effect of Akt.

  At low magnification (4x) used for population-based analysis, about 50 to 500 neurons per field (e.g., about 100 to about 400, about 150 to 350, or about 300 per field) Of neurons) was observed. Single cell-based analysis is performed at a higher magnification (20x), which is the magnification that allows spatial resolution of changes in neurons (e.g., inclusion body formation, dendritic shape changes, etc.). At this magnification, about 10 to 100 neurons (about 15 to about 75, about 20 to 50, or about 30 neurons per field) could be observed. Single cell-based analysis was performed in this example by analyzing all neurons in three random images that were tracked over time (i.e., acquired a predetermined number of times over a predetermined selected period). did. It is justified to apply Kaplan-Meier analysis (this analysis is basically a long-term analysis for an individual subject), which returns to the same field of view of the microscope each time and disappears in the time to return. This is because it “guesses” the number of nerve cells.

  Automated analysis was used to first select a specific field and monitor the number of neurons in the field at each measurement interval. Since these neurons were post-mitotic, the number of neurons in the visual field at a particular point in time was subtracted from the number of previous neurons in that visual field to kill the neurons that died at some point in the intervening period. The number of cells was estimated. For the purposes of survival analysis and convenience, for neurons that died during the non-observation period, the time from the transfection to the first disappearance from the image was defined as the time at which cell death occurred.

Although the invention has been described with reference to particular embodiments, those skilled in the art will recognize that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Should do. Those skilled in the art will appreciate that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Many modifications can be made to adapt a particular situation, material, composition of matter, method, and process steps to the objective, spirit, and scope of the present invention. All such modifications are within the scope of the claims appended hereto. The scope of the invention is limited only by the literal or fair scope of the following claims.

Furthermore, the inventor has also found that the variation pattern of gene expression is different among a plurality of cells cultured in the same petri dish. Further, according to the optical condition investigated by the inventor, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) of the objective lens of the imaging apparatus is 0.071 or more. It was found that a cell image that can be imaged within 1 to 5 minutes and can be analyzed is provided. A light emission analysis system that observes these light emission images with a storage-type imaging device under a microscope is called a light emission microscope. The light emission microscope preferably has a light shielding means having an opening / closing lid (or an opening / closing window) for shielding light, and a necessary biological sample is set or exchanged by opening / closing the light shielding means. Depending on the purpose, an operation for chemically or physically stimulating a vessel containing a biological sample may be performed manually or automatically. In the best mode, the luminescence microscope is equipped with a known or unique culture device. The culture apparatus has a function of optimally maintaining the temperature, humidity, pH, outside air component, medium component, and culture solution component so as to enable analysis in the system for a long period of time.

  Examples of the biological material from which the luminescent sample is derived include cells or tissues derived from eukaryotes and cyanobacteria. In medical use, a sample containing cells excised by biopsy from a site to be examined in mammals, especially humans is particularly exemplified. In regenerative medicine, at least a part of an artificially modified or synthesized biological sample can be used for the purpose of examining whether biological activity is maintained well. In another aspect, the assay subject of the present invention is not limited to cells or biological tissues derived from animals, but may be cells or biological tissues derived from plants or insects. In the case of bacteria and viruses, the analysis of each part in the container that has not been performed by a conventional luminometer can be targeted. In a luminometer, an infinite number of samples (for example, 1 million or more per well) are stacked in a container such as a well or a petri dish to quickly obtain a strong light emission amount. In the present invention, since an image of each luminescent sample that cannot be seen with the naked eye is generated, individual cells or biological tissues can be analyzed even if they are contained in a container at a density that allows individual cells to be identified. Such individual analysis includes analysis methods in which only luminescent cells are statistically summed or averaged. Thereby, the evaluation regarding the exact interaction per cell can be performed. In addition, in a large number of mixed luminescent samples, it becomes possible to identify cell groups or tissue regions having the same luminescence amount or luminescence pattern.

Supplementary explanations of terms relating to the first embodiment and modified sample Sample containers containing the sample 1 include a petri dish, a slide glass, a microplate, and a flow cell. Here, the container bottom is a container having a wide (or flat) bottom so that two-dimensional data can be easily obtained as well as a light-transmitting material (glass, plastic, etc.). Is more preferable. In a well or cuvette in which a plurality of storage portions are integrated, it is preferable that the partition portion of each storage portion is formed entirely with a light-shielding material or dye. In addition, for containers having an upper opening such as a petri dish, it is preferable to cover the evaporation prevention lid, and the inner surface of the lid is coated with an antireflection coating or dyeing in order to improve the S / N ratio. preferable. Instead of such a hard lid, a liquid lid such as mineral oil may be arranged on the upper surface of the sample in the container. The sample stage on which the sample container is placed may be moved in the X-axis direction and the Y-axis direction in order to change to another field of view for imaging as required by a general microscope device. Good.

  The objective lens 2 may be arranged in an inverted form below the sample 1. The objective lens 2 may be heated by appropriate heating means (such as a Peltier element or a warm air heater) so that the living luminescent sample functions stably in a constant temperature environment such as culture conditions. The objective lens 2 may be further driven in the Z-axis direction that is the optical axis direction (vertical direction in the figure). A mechanism 9 is provided to automatically drive the objective lens 15 along the Z-axis (optical axis direction). Examples of the Z-axis drive mechanism of the objective lens include a rack and pinion mechanism and a friction roller mechanism.

  Further, the objective lens 2 can be appropriately oil-immersed according to a desired magnification. Which magnification is selected is arbitrarily set according to the size of the sample to be evaluated (or analyzed). Specifically, the magnification can be low enough to observe cells and tissues (for example, 5 to 20 times) or high enough to observe minute substances inside or outside the cells (for example, 40 to 100 times). .

  The monitor 5 is preferably provided with an analysis method for observing a change in activity relating to one or more desired cells with a real-time image by providing a configuration in which image information of the luminescent sample is displayed as a moving image. . Thereby, it becomes possible to observe the state of light emission in the cell unit or the tissue unit in time series with a realistic image.

  In the present invention, the method and apparatus for observing in time series may be provided in the form of software for controlling or coordinating the required apparatus configuration or a computer program characterizing the software. In addition, by electrically connecting the method or apparatus of the present invention to a database arranged identically or separately from the apparatus, it is fast and reliable and high quality without being limited by image capacity or analysis information amount. Analysis results can be provided.

In the present invention, by using light emission (cold light) from a substrate solution as a chemical excitation reagent, a configuration for irradiation with excitation light is not required in the detection step. Furthermore, in observation using a microscope, a cryogenic temperature (for example, −30 to −60 ° C.) using liquid nitrogen is obtained by using an objective lens having a high numerical aperture for a luminescent sample that emits weak light that cannot be seen with the naked eye. It is possible to perform high-speed imaging without employing a large-sized and high-cost super-cooled CCD that requires cooling. Specifically, an objective lens having a high numerical aperture (high NA) and a projection magnification that includes an object in the observation field, and a low temperature (for example, a high temperature of −5 ° C. or higher) at room temperature ( Or an image sensor (CCD, CMOS, etc.) that can function continuously in the vicinity of the temperature within the apparatus), holding means for holding the object in a position suitable for image generation, and these objective lens, image sensor, and holding means. A light shielding means for ensuring light shielding at the time of image generation is provided as a main component. When the CCD is small and can be controlled at a low temperature, it is compact as a total system, and even if all the components are housed in the same housing, it can be designed to have a height that can be looked down on a desk. Therefore, an apparatus embodying the present invention is a very small and low cost product. By downsizing, the space inside the device also becomes small, so it becomes easier to adjust the conditions of the biological environment (temperature, humidity, air components) in the device to a level suitable for culturing and trace reactions, further reducing costs There is also an advantage of having high reliability. In addition, by reducing the overall height of the device, it is possible to easily perform stimulation processing such as taking in and out of luminescent samples and dispensing of chemicals to the stored samples from the top of the device through appropriate opening / closing windows or opening / closing covers. Yes.

  On the other hand, the present invention provides a method and apparatus for generating an image with a large number of pixels (preferably a large number of pixels) without performing optical scanning. In the image analysis of the generated image, image analysis software for analyzing cells can be provided as a part of the present invention, which can be used industrially as a single system or as a total system with a light emission microscope.

The following description regarding the high-throughput imaging apparatus is also included as a single aspect of the present invention or in combination with the invention described in the above description. However, other methods or apparatuses that can share the following contents are not limited to the gist of the present invention within the scope of the following description, and include descriptions of dividable inventions.

Technical field of high-throughput imaging equipment
The present invention relates to an imaging apparatus that images a large number of imaging regions set in one or more biological samples by time lapse, and more particularly to a high-throughput imaging apparatus that enables high-throughput imaging. The present invention also relates to software including a program for performing the function of such an imaging apparatus. The present invention is suitable for research or clinical use in which many biological activities of biological samples such as cells derived from living organisms are efficiently examined.

Background art of high-throughput imaging devices Since organisms have a high degree of complexity, it is not easy to understand the structure and function. For this reason, in recent years, a simple experimental system using cells that are the smallest unit capable of reproducing life phenomena, that is, cultured cells, has been used. By using cultured cells, it is possible to conduct experiments in which the analysis of hormone responses and the like is not affected by other factors in the living body. In other words, it is possible to analyze the function of a gene by introducing or inhibiting the gene.

  In order to culture cells, it is necessary to use an environment that mimics the living body. Therefore, the temperature is set to 37 ° C., which is a body temperature, and a medium simulating an intercellular fluid is used. In addition to nutrient sources such as amino acids, the medium contains a carbonate buffer for pH adjustment. The carbonate buffer is in an equilibrium state in the presence of air containing carbon dioxide with a partial pressure as high as 5%, and is used for culture in an open system such as a dish. Moreover, in order to prevent water from evaporating from the culture medium, a high humidity environment of 95 to 100% is required.

  A carbon dioxide incubator having the above environmental conditions is used for cell culture. Furthermore, a phase contrast microscope, a differential interference microscope, and a fluorescence microscope are used for expression observation of GFP for cell state observation. In addition, a CCD camera and a controller (personal computer) are used for taking and displaying a microscope image, and a culture microscope in which these are combined has been proposed (Patent Document: JP 2003-29164 A).

When observing cells cultured in a long-term or long-term with a microscope for problems to be solved related to the high-throughput imaging apparatus, images are acquired in a time series using a time-lapse observation method. Time lapse is used to make it easy to check the state of cells that change over a long period of time by taking a sample and storing an image at regular intervals. For example, if one cell is first photographed for the required photographing time (camera exposure time) and then taken once every hour for 24 hours, 25 images can be acquired. If these are photographed and then played back continuously, changes in cells every hour can be easily confirmed. If the imaging interval is shortened to 30 minutes or 15 minutes, for example, fast-moving cells can be observed.

  When it is desired to observe a plurality of cells, the microscope or sample is moved to a target location using an electric stage attached to the microscope. The movement to the observation position is performed in synchronization with the time lapse. Such a time lapse for sequentially observing a plurality of observation positions is called a multi-point time lapse. In addition, according to the light emission imaging method and the light emission imaging device of the present invention described above, by adopting an objective lens with a bright numerical aperture that has been uniquely investigated, a significantly shorter time (for example, 1/30 or less) than in the past. The time-series evaluation can be realized with a weak light image in units of 5 to 20 minutes, less than 5 minutes under suitable conditions, and about 1 minute at the shortest. This makes it possible to replace or link a luminescent image that cannot be seen with the naked eye to a fluorescent image with a luminance that can be observed with the naked eye, and also to analyze the dynamics of biomolecules at high speed. It provides a breakthrough technology that can be used. In order to obtain a luminescent image, no excitation light is required, so of course not only photosensitivity tests such as circadian rhythm, but also accurate and long-term analysis without excessive stimulation or damage to biological materials. . In regenerative medicine, there is a possibility that medical use such as treatment, diagnosis, drug discovery, etc. using a biomaterial that has not received any biological damage is possible by analysis using a luminescent image.

However, when imaging by time lapse is performed for a large number of cells, there is a case where there is a significant time difference between the first imaged cell and the last imaged cell. Significant time differences are often fatal when making cell-to-cell comparisons. In addition, an effective method for imaging so as not to cause a time difference between a plurality of arbitrarily designated cells is not known. Conventionally, there is only known a method of manually changing the imaging interval so that a plurality of cells are sequentially imaged, and stopping and retrying a malfunction. In particular, in a system that automatically analyzes a large number of cells, such as a recent cell-based assay, there is a possibility that the throughput may be drastically reduced by using time-lapse images.

  In view of the above circumstances, an application example in which the method and apparatus of the present invention are improved so as to efficiently perform time-lapse shooting work will be described below. Note that the high-throughput imaging apparatus and software therefor described below are excellent inventions having further patentability based on the gist of the present invention. Here, an object of the invention shown below can be to provide a high-throughput imaging apparatus capable of obtaining time-lapse data from a large number of imaging targets at the same time and software for the same.

In order to achieve the above object, the high-throughput imaging apparatus according to the present invention captures a biological sample existing in a plurality of imaging regions and obtains a sample image. An image acquisition unit for controlling the image acquisition unit, and a control unit for controlling the image acquisition unit and performing time-lapse interval imaging for each imaging region, and the control unit includes an imaging time required for acquiring the sample image And interval imaging condition setting means for setting an interval imaging condition based on the number of imaging areas. As an example of the conditions for interval imaging, changing the imaging time as the time-lapse condition according to the activity rate or reaction rate of the biological sample is also included. According to the light emission imaging method and apparatus of the present invention, an appropriate exposure time can be selected from an exposure time of 1 minute to 20 minutes or longer. For a sample with a fast reaction (or activity) speed, the minimum exposure time can be set within a range where a luminescent image at a level that allows image analysis is obtained. Conversely, for a sample with a slow reaction (or activity) rate, a long exposure time can be set to such an extent that the image capacity is not excessive. Preferably, in the same or different field of view, a plurality of exposure times (for example, any combination of two or more from the group of 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes) or continuous time ranges ( For example, in an exposure time of 1 to 30 minutes, by setting a division scale in units of 1 to 3 minutes or an arbitrary exposure time selected in a stepless manner, for each sample or for each region (or part) in the sample Perform image analysis. Furthermore, by controlling the playback speed of the image playback means for the luminescent images with different reaction (or activity) speeds, it is possible to provide a moving image with a pseudo-similar speed, making it easy and efficient to evaluate the diagnosis and the like. Also, there is an advantage that the final evaluation result can be obtained quickly because accurate analysis can be performed even when there is non-specific variation for each sample. In addition, a minimum exposure time can be set within a range where a light emission image at a level that allows image analysis can be obtained for each sample, which helps shorten analysis time and increases throughput. The exposure time may be selected automatically or manually.

Further, the high-throughput imaging apparatus of the present invention is an image acquisition means for acquiring a sample image by imaging a biological sample existing in a plurality of imaging regions by an image information extraction means for extracting different image-related information. And control means for controlling the image acquisition means and performing time-lapse interval imaging for each imaging region, the control means including an imaging time required for acquiring the sample image and the image information extraction means It is characterized by having interval imaging condition setting means for setting the conditions for interval imaging based on the type of the above. Even in such a configuration, since the imaging operation is efficiently executed according to the imaging conditions set by the control means, it is possible to image a large number of imaging regions in a short time.

In the present invention, a “biological sample” can be any living organism, and can be held in a container or living organism as a suitable holding means in a state where the biological activity of interest can be maintained. An imaging region to be imaged can be provided to the imaging means. Here, the container includes any container that holds a sample in a state where imaging can be performed by a desired imaging unit, and specifically includes a well, a petri dish, a slide chamber, and a cuvette. Examples of living organisms include plants, mammals, fish, insects, bacteria, and viruses. When the life form is maintaining life, if necessary, it is necessary to treat a part of the life form so that it can be imaged. The sample is held by the living organism. A biological sample includes any part derived from a living organism, but is preferably a biological cell, more preferably a nucleated cell capable of developmental division or proliferation. For an organism that has built up multiple organs in which cells are separated into different functions, it may be any organ that exhibits the biological activity of interest. The biological activity can be one or more of physiological, genetic, immunological, biochemical, and hematological activities. The “plurality of imaging regions” means one or more biological samples held in the same holding means, or one or more types of imaging sites held in separate holding means.

As described above, the high-throughput imaging apparatus according to the present invention efficiently performs the imaging operation according to the imaging conditions set by the control unit, and thus can capture a large number of imaging areas in a short time. It becomes possible to do. In addition, since a large amount of time-lapse data can be obtained at the same time even when the number of samples is very large, it greatly contributes to research and clinical studies on biological activity.

  A first embodiment of the present invention will be described with reference to FIG. FIG. 23 is a conceptual diagram showing the overall configuration of the apparatus of the present invention. In the culture microscope main body 101, an incubator chamber for culturing cells and a microscope portion for observing the cells are integrated. The culture microscope main body 101 incorporates a controller 102 to control each unit to be described later. The controller 102 is disposed inside the culture microscope main body 101 in order to make the space of the culture microscope compact. However, the controller 102 may be disposed outside the main body when there is an influence of heat generated by the controller. Furthermore, the culture microscope main body 101 includes a warning buzzer 103 and a warning display device 104. The warning buzzer 103 can beep when a problem occurs during the experiment. In addition, the warning display device 104 can display a warning when a problem occurs, a work instruction, and the like, like the warning buzzer 103. In particular, the warning display device 104 is a touch panel 104 a having a function as an operation panel, and an operator can select an operation by touching the touch panel in accordance with an instruction displayed on the warning display device 104.

  A focus handle / joystick 105 is connected to the controller 102, and the R stage and the θ stage can be moved with the focus handle in the Z-axis direction of the microscope portion, which will be described later, that is, the direction to focus on the sample and the joystick. it can. The θ stage is an electric stage that can move in the direction of rotation about an axis, and the R stage can move in the direction of one axis perpendicular to the central axis of the θ stage. These are used to reduce the size of the apparatus, but a general XY stage may be used. In particular, the R stage motor 30 and the θ stage motor 31 are driven and controlled through the controller 102 based on conditions set by interval imaging condition setting means of the apparatus of the present invention described later.

  The culture microscope main body 101 has a heater 112 for temperature control in the incubator chamber, and a temperature controller 106 for controlling the heater 112 is provided.

  The controller 102 and the temperature controller 106 are connected to a computer 109 (a personal computer in FIG. 23) through an interface such as RS-232C, and can be controlled from the computer 109. Each means necessary for an interface (memory, arithmetic circuit, display unit, input unit, etc.) is also included.

  Temperature (37 ° C.), humidity (95-100%), carbon dioxide (carbon dioxide; 5%) (each numerical value is a general value and can be adjusted) in the incubator chamber of the culture microscope body 101 There is a tank 107 in which mixed air is controlled, and the mixed air can be supplied by opening and closing the electromagnetic valve 108. In the present invention, mixed air is put into the tank 107, but the tank 107 may be made of only carbon dioxide, and a water tank (not shown) for maintaining humidity may be installed in the incubator chamber. Further, it is possible to supply carbon dioxide gas to the incubator chamber without maintaining the tank 107 at 37 ° C. This electromagnetic valve 108 may be controlled by a computer 109.

The computer 109 is connected to a network 110 such as a LAN or the Internet. Further, the network 110 is connected to a remote computer 111, and the computer 109 is controlled from the remote computer 111 (a personal computer in FIG. 23) via the network 110. Accordingly, the culture microscope main body 1 can function as a database capable of monitoring time-lapse interval imaging from the remote computer 111 or storing a large amount of imaging data for retrieval. The remote computer 111 may be a stakeholder of the user, but it is preferable to cover it with a business contract with an external specialist who operates the system in order to continue the use of time-lapse imaging over a wide variety of complex. In addition, if the remote computer 111 is a place other than the site of the apparatus (examination room, laboratory, etc.), it is mainly intended for monitoring at any time of leaving the room, returning home, claiming, or vacation time. It may be a portable image receiver. According to the portable remote computer 111, it is possible to immediately respond to calling a desired image or grasping an abnormality with a buzzer, a lamp, an abnormality mark or the like at the time of warning. In any case, in the remote computer 111 connected to the apparatus main body by communication means, access by appropriate authentication means (for example, password, person ID, electronic key, biometrics (fingerprint, iris, vein, etc.)) It is preferable to limit. Further, it is more preferable that an access person who has been appropriately authenticated can be remotely controlled to change the interval imaging condition through the remote computer 111. In this way, the remote computer 111 can also enable monitoring and some operations without going to the site of the device, greatly reducing the burden on the user (eg, man-hours, expenses, travel time). Provide an excellent usage system.

  FIG. 24 is an internal configuration diagram of the culture microscope main body 1 of the present invention. The incubator chamber 20 is sealed from the outside by a lid 22, and maintains the temperature, humidity, and carbon dioxide (CO2) concentration of the culture environment inside it, and actively controls them. The mixed air is supplied from the tank 7 via the air pipe 24. Unnecessary air is discarded from a pipe (not shown). The lid 22 can be opened and closed by a handle 21 around a hinge 23. When the lid 22 is open, the lid open / close sensor 28 operates to notify the controller 2 of the opening / closing of the lid 22.

The heater 12 is installed in the incubator chamber 20 and can automatically operate and maintain the temperature when it detects that the temperature is not higher than 37 ° C., for example, by a temperature sensor (not shown). Although only one heater 12 is shown in FIG. 24, the temperature unevenness in the incubator chamber may be reduced by being attached to the entire lid 22 and base 55.

  The circular tray 26 has a plurality of sample installation holes 52, in which a plurality of sample containers 25 can be installed. The sample container 25 can be taken out upward with respect to the circular tray 26. On the other hand, when the sample container 25 is installed on the circular tray 26, the bottom surface of the sample container 25 contacts the ring-shaped protrusion 51 of the sample installation hole 52 of the circular tray 26 so that it does not fall down. Further, the sample container 25 can be positioned with respect to the circular tray 26 and can be installed. The bottom surface of the sample container 25 is made of transparent glass or resin and can be observed from the objective lens 33.

  In addition, the sample container lid 57 may cause condensation when the sample is taken out of the incubator chamber 20 by exchanging the medium and placed in the incubator chamber 20 with the sample cooled. A space for storing as a spare is provided so that the sample container lid 57 can be replaced when the condensation occurs. Since the sample container lid 57 is in the incubator chamber 20 even during medium exchange, it does not cool. Further, the sample container 25 is made of a transparent and observable bottom member 93 such as glass and a top member 91 having members 90 and 92 having a large heat capacity such as metal, and the bottom member 93 and the member 92 are bonded together. The upper surface member 91 is bonded to the member 90. The member 92 and the member 90 are detachable. With such a structure, dew condensation between the top member 91 and the bottom member 93 is prevented.

  The circular tray 26 can be detached from the rotating base 34. When the circular tray 26 is removed, the circular tray attachment / detachment sensor 27 is activated to notify the controller 2 that the circular tray 26 has been removed. The circular tray attachment / detachment sensor 27 is shown as a push button type in FIG. 24, but any sensor that can detect attachment / detachment of the circular tray 26 may be used.

The rotation base 34 is attached to a θ rotation shaft 35, and the rotation of the θ stage motor 31 can intermittently stop the rotation of the circular tray 26 one tray at a time in the direction of the arrow in FIG. The rotation period composed of the rotation and stop of the circular tray 26 is driven and controlled through the controller 2 based on the conditions set by the interval imaging condition setting means of the apparatus of the present invention described later. As a variation of the rotation cycle, by performing a rotation stop cycle that stops at a rotation distance that is one tray more or less than the number of all sample containers 25 arranged on the same circumference on the circular tray 26, Although it appears to stop intermittently by one sample container, it is possible to make almost all the sample containers 25 one turn each time they move intermittently. It is also possible to monitor the quality of the culture state relating to 25.

  The lead screw 38 is rotated by the R stage motor 30, and the linear movement base 36 attached to the nut 53 moves to the left and right. The linear movement base 36 has a linear guide 54 that can move only in the linear direction. The θ rotation shaft 35 is rotatably attached to the linear movement base 36. When the linear movement base 36 moves to the left and right, the rotation base 34 can also move to the left and right. Thereby, the stage which can move a sample by a R (theta) polar coordinate system is realizable.

  The base 55 separates the incubator chamber 20 and the motor chamber 58, and each part is sealed so that high-humidity air does not enter the motor chamber 58. First, a planar sheet 50 is sandwiched between the rotating base 34 and the base 55 so as to be slidable.

  The bellows 56 is attached so as to surround a portion where the objective lens 33 is exposed in the incubator chamber 20, and is sealed with an end face fixed to the front end portion of the objective lens and the base by bonding or the like. This prevents high-humidity air from entering the motor chamber 58 through the gap between the base 55 and the objective lens 33.

  The objective lens 33 can be moved up and down by the Z stage motor 32 turning the lead screw 39. The objective lens 33 moves up and down to focus on the sample. Even if the objective lens 33 is moved up and down, the bellows 56 is made of a soft resin such as rubber, so that it can be expanded and contracted, and hermeticity is maintained.

  The microscope chamber 59 is designed to maintain the temperature to such an extent that the optical system member does not expand due to a temperature change. A heater or the like (not shown) is used for maintaining the temperature.

  A controller 2 is installed in the microscope room, and wiring is connected to each unit. The LED illumination 41 is illumination for fluorescence observation, and illuminates the sample via the fluorescent cube 42 and the passing window 40 and the objective lens 33. Light from the sample passes through the objective lens 33, the passing window 40, and the fluorescent cube 42, passes through the magnification changing lens 43, and is incident on the CCD camera 45 with the optical path bent by 90 degrees. The mirror 44 is provided to secure the installation space for the CCD camera 45. If there is an installation space for the CCD camera 45, there is no need to bend the optical path.

  Instead of the LED illumination 41, an optical fiber such as a mercury lamp (not shown) can be used as a light source. In the case of a mercury lamp, since it cannot be turned on and off at high speed like the LED illumination 41, it is necessary to attach a shutter to turn on / off the incidence of light. These can also be controlled from the controller 2. In some cases, the light enters the CCD camera 45 without passing through the magnification changing lens 43. That is, the magnification changing lens 43 may be appropriately inserted into and removed from the optical path extending from the objective lens 33 to the CCD camera 45.

  The fluorescent cube 42 is rotatable around an axis 48, and can be switched to fluorescent cubes having different wavelengths. The rotation can be performed electrically by driving a cube turret motor 47. These can also be controlled from the controller 2.

  The magnification changing lens 43 is rotatable around an axis 49, and can be switched to a lens having a different magnification. The rotation can be performed electrically by driving the lens turret motor 46. These can also be controlled from the controller 2. The magnification changing lens 43 may be a single zoom lens with a built-in zoom function. Moreover, the structure which only replaces | exchanges for the lens of specifications, such as desired magnification, as needed may be sufficient.

  FIG. 25 is a block diagram showing units that can be controlled by an electrical method or the like in the units shown in FIGS. Although the description of each unit described in FIGS. 23 and 24 is omitted, each unit is connected to the controller 102 and can be controlled by the observer from the user interface of the computer (personal computer) 109. The CCD camera 45 is a high sensitivity type using a cooled CCD and is directly connected to the computer 109. The heater 112 is connected to the computer 109 via the temperature controller 106, but if the controller 102 has the function of the temperature controller 106, the heater 112 may be controlled via the controller 102.

  As a characteristic configuration of FIG. 25, in the external control system 60 that controls the function inside the apparatus main body 101 from the outside, the interval imaging condition setting means 70 that sets the imaging condition for each set sample container 25 uses the computer 109. It is connected to the controller 102 via this. Another feature is that the controller 102 is connected so as to drive and control the imaging operation of the CCD camera 45 as the imaging means. Thus, the external control system 60 including various interfaces such as input means and display means attached to the computer 109, the interval imaging condition setting means 70, the controller 102 as the internal control system, and the CCD camera 45 as the imaging means. And various motors 30 and 31 for the circular tray 26 holding each sample container 25 can be linked.

Here, the setting contents by the interval imaging condition setting means 70 will be described. The interval imaging condition setting means 70 provides a device that can cope with assumptions in every scene as shown below.
Apparatus 1: Image acquisition means for acquiring an image of a biological sample existing in a plurality of imaging areas and controlling the image acquisition means, and time lapse interval imaging for each imaging area Control means for performing an interval imaging condition for setting an interval imaging condition for each imaging area based on an imaging time required to acquire the sample image and the number of imaging areas A high-throughput imaging apparatus comprising a setting unit.
Apparatus 2: The high-throughput imaging apparatus according to the apparatus described in the apparatus 1, wherein the interval imaging condition has a setting for switching a plurality of imaging areas a plurality of times to perform imaging.
Apparatus 3: The high-throughput imaging apparatus according to the apparatus described in the apparatus 2, wherein the image signals sent from the image acquisition unit are integrated for each imaging region to generate an image.
Apparatus 4: The high-throughput imaging apparatus according to the apparatus described in the apparatus 1, wherein the interval shooting condition includes a surplus time for performing another type of processing in addition to the time required for imaging.
Apparatus 5: In the apparatus according to the apparatus 4, in order to maintain the imaging region in an optical environment that can be imaged by the image acquisition unit, a light shielding unit that shields light from the outside and a light environment by the light shielding unit are temporarily A high-throughput imaging apparatus comprising: processing means for canceling and performing processing other than imaging by the image acquisition means.

Apparatus 6: The high-throughput imaging apparatus according to any one of the apparatuses 1 to 5, further comprising a moving unit that moves the image acquisition unit relative to the plurality of imaging regions.
Device 7: In the device described in the device 6, a plurality of biological samples are sequentially arranged along the circumference of the circular tray, and the circular tray can be rotated and stopped according to the imaging conditions of the control means. A high-throughput imaging apparatus.
Apparatus 8: The high-throughput imaging apparatus according to the apparatus described in the apparatus 7, wherein the rotation period of the circular tray is stopped after passing through a plurality of imaging regions.
Apparatus 9: The apparatus according to the apparatus 8, wherein the rotation period of the circular tray has a long-distance rotation mode corresponding to an imaging area of 1 rotation ± 1.
Device 10: The device according to Device 9, further comprising reference information acquisition means for acquiring different types of reference information regarding all imaging regions on the circular tray during long-distance rotation in the long-distance rotation mode. High-throughput imaging device.
Apparatus 11: In the apparatus according to any one of the apparatuses 1 to 4, the image acquisition unit includes an imaging magnification changing unit capable of changing an imaging magnification at the time of imaging, and an interval according to an imaging magnification by the imaging magnification changing unit. A high-throughput imaging apparatus characterized by setting imaging conditions.
Apparatus 12: In the apparatus according to any one of the apparatuses 1 to 4, the image acquisition unit includes a light receiving element that receives an optical signal obtained from the imaging region, and sets an interval imaging condition according to the light receiving capability of the light receiving element. A high-throughput imaging apparatus.

Apparatus 13: An image acquisition means for acquiring an image of a biological sample existing in a plurality of imaging regions by an image information extraction means for extracting different image related information, and controlling the image acquisition means And control means for performing time-lapse / interval imaging for each imaging area, wherein the control means is based on the imaging time required to acquire the sample image and the type of the image information extraction means. A high-throughput imaging apparatus comprising interval imaging condition setting means for setting conditions for interval imaging for each.
Apparatus 14: The high-throughput imaging apparatus according to the apparatus described in the apparatus 13, wherein the image information extraction unit extracts different pieces of image related information simultaneously or continuously with respect to the same imaging area.
Apparatus 15: The high-throughput imaging apparatus according to the apparatus described in the apparatus 14, further comprising image synthesis means for synthesizing different image related information extracted by the image information extraction means in association with a sample on an imaging region.
Device 16: The device according to any one of the devices 13 to 16, wherein the image information extraction means is a combination of two or more sets of transmitted light, fluorescence, bioluminescence, chemiluminescence, Raman spectroscopy, and infrared rays. High-throughput imaging device.
Apparatus 17: The apparatus according to any one of the apparatuses 1 to 16, further comprising a culture means for continuously culturing cells in the biological sample, and the cells existing in the imaging region to which the control means returns A high-throughput imaging apparatus, characterized in that interval imaging conditions are set according to imaging timing during each culture period.

  FIG. 26 is a top view of the circular tray. In the circular tray 26, the sample setting holes 52 into which the sample containers 25 are placed are distributed at equal intervals on the circumference around the axis of the θ rotation shaft 35. The sample information regarding the sample container 25 set in each sample installation hole 52 is stored in a built-in memory in the computer 109, and is called when setting the time-lapse interval imaging condition, and the imaging condition is determined and the image is captured by the controller 102. Executed. Information on the determined imaging condition is notified to the computer 9 via the controller 102, stored in the memory of the computer 9 in association with the sample information, and can be displayed on the user interface as appropriate.

The user interface of the computer 9 is given an individual ID of the sample container 25 of the circular tray 26, and is programmed so that the operations of the imaging device and the circular tray correspond to each other through this ID. The user interface input means (optical mouse, keyboard, touch panel, electronic pen, etc.) prompts the computer 9 to set imaging conditions based on information selected by the user for each ID. Here, as shown in the flowchart of FIG. 27, the observation preparation state is set, a GUI is displayed (S1), and the stage origin search is executed (S2). Here, after the user can input the sample container to be observed and waits for input (S3), preferably press the direction arrow buttons in Stage / Rθ and Stage / Z to display the cell image in the sample container in the live image window. As shown in FIG. 4, a cell for which time-lapse imaging is desired is searched, and the position is selected on the display screen by the input means. That is, in this example, first, a bright field image and / or a luminescent image as described above is displayed, and then a desired cell, a cell group, a tissue region, and a specific site in the cell are designated. Yes. Preferably, it is preferable to display the current imaging result or analysis result at any imaging time, even during the time-lapse imaging. For this purpose, it is more preferable to execute control so as to obtain a quick result by sequentially analyzing the light emission images obtained during the time-lapse imaging.

  Next, the observation condition related to the selected observation position is waited (S4), and the user inputs a desired observation condition (S5). In the input, for example, observation conditions that meet the requirements (for example, the wavelength of weak emission, detection sensitivity, brightness in bright field observation, etc. depending on the type of reagent used and experimental conditions) are input in the same manner as described above. Moreover, in the example which uses fluorescence measurement together as shown in the figure, for example, it is selected whether to use LED-G (green) or LED-B (blue), or the brightness of the LED illumination 41 is determined. In addition, the fluorescent cube corresponding to the wavelength selected by Cube is selected, or the magnification changing lens corresponding to the numbered button is determined by Lens. In addition, the camera shooting conditions such as the exposure time of the CCD camera and whether or not to execute AE are determined with Camera Control, the file name for saving the captured image is determined with Image File Name, and the time lapse is determined with Time-lapse. All necessary parameters are set as observation conditions, such as setting the interval time and experiment period. Here, the time lapse interval time refers to the electric stage moving time, photographing time and control time for photographing the first multi-point when performing multi-point time lapse (including the case of only one point), the multi-point The total waiting time until immediately before the second shooting is started.

Next, when the inputted shooting conditions are stored (S6), for example, when the total time of the stage movement time and the camera exposure time is longer than the time lapse interval time, time lapse interval imaging cannot be performed correctly. Therefore, the computer can reset the interval time of the time lapse and reset the imaging time so that a desired number of imaging areas can be imaged without omission. For example, when an automatic adjustment button is clicked, a time slightly longer than the total time of stage movement time and camera exposure time can be automatically calculated and set as the time lapse interval time. Alternatively, the time may be switched to continuous time-lapse imaging by bringing the standby time close to zero. Thus, when the setting at the specific observation position is completed (S7), the preparation for observation is completed, and imaging can be started. If it is desired to cancel the storage of the input condition and input again, or to set another observation position, the process returns to S3 and the input is repeated.
As described above, according to the multipoint time lapse shown in this example, it is possible to automatically observe each desired observation target with high throughput and appropriate imaging conditions without omission.

  As described above, in the above example, the example using the fluorescence microscope has been described. However, the present invention may capture the emission image exclusively, and in that case, it is not necessary to irradiate the excitation light. The system can be removed. Observation of fluorescently labeled cells or the like may be performed simultaneously or separately with images of luminescently labeled cells or the like. Examples of luminescent labels include bioluminescence (or chemiluminescence) that emits faint light that cannot be seen with the naked eye even under a microscope.

   Examples of bioluminescence (or chemiluminescence) include cells or tissues into which DNA containing a luciferase gene is introduced as a reporter gene linked downstream of a promoter of a gene region of interest. By using a cell or tissue in which luciferase is expressed as a reporter, it is possible to detect changes in transcription over time by detecting luciferase activity at a desired expression site.

  A preferred embodiment is a vertebrate cell or tissue in which the introduced luminescent gene is expressed in a rhythm in peripheral tissues. Peripheral tissues include, but are not limited to, liver, lung, and skeletal muscle. These peripheral tissues are reported to have a circadian rhythm with a phase difference of 7-12 hours. The delay pattern of circadian rhythm is thought to reflect the normal coordination of the biological rhythm of complex mammals composed of multiple organs.

  According to this, the information analyzed according to the present invention is intended to elucidate the mechanism of jet lag or sleep disorder related to circadian rhythm, and to screen and test compounds useful for the treatment of circadian rhythm disorder. It can be said that it is useful for developing a mammal model to be used as

  Moreover, various tests or screenings can be performed by using a transformant or a transgenic mammal containing the DNA of the present invention that expresses a reporter gene. By detecting reporter gene expression in these tissues or cells under a variety of arbitrary conditions, it is possible to assess or screen for the effects of stimuli or compounds that modulate reporter gene expression . Stimulation includes temperature, light, exercise, and other shocks. There are no restrictions on the compounds used. In particular, the present invention relates to a compound that modifies the expression induced by the promoter of a clock gene (for example, Period 1) introduced into the transformant or transgenic mammal of the present invention, and the transformant or transgenic mammal. It is applicable to the method of using or testing.

Examples of the test or screening method of the present invention include the following methods.
Method (1): A method for testing or screening a compound having an activity of altering the expression of a transgene in the transformant of the present invention, comprising: (a) treating the transformant with the compound; b) measuring the expression of the transgene in the treated transformant.
Method (2): A method for testing or screening a compound having an activity of altering transgene expression in a mammal of the present invention, comprising: (a) treating the mammal with the compound; and (b) Measuring the expression of the transgene in the treated mammal.

The method of the present invention is useful for screening compounds that modulate the expression of Period 1 gene. The method is also useful for screening pharmaceuticals for circadian rhythm disorders. Therefore, in addition to the above methods (1) and (2), the following screening methods are also possible according to the present invention.
Method (3): A method for testing or screening a pharmaceutical agent useful for the treatment of circadian rhythm sleep disorder, wherein (a) treating the transformant or transgenic non-human mammal of the present invention with the pharmaceutical agent; And (b) measuring the expression of a reporter gene in the treated transformant or mammal.

  There is no particular limitation on the compound used in the test or screening method of the present invention. Examples include inorganic compounds, organic compounds, peptides, proteins, natural or synthetic low molecular compounds, natural or synthetic high molecular compounds, tissue or cell extracts, microbial culture supernatants, plants or marine organisms. Natural ingredients derived from, etc. are included, but are not limited thereto. Expression products such as gene libraries or cDNA expression libraries may be used. There is no particular limitation on the method of treatment with the compound. In vitro treatment may be performed, for example, by adding the compound to the culture medium and contacting the cell with the compound, or introducing the compound into the cell using a microinjection or transfection reagent. Methods of treatment in vivo include intraarterial injection, intravenous injection, subcutaneous injection, or intraperitoneal injection; oral administration, enteral administration, intramuscular administration, or intranasal administration; ocular administration; injection or catheterization. Methods known to those skilled in the art, such as intracerebral administration, intraventricular administration, or peripheral organ administration, are included. The compound is suitably administered as a composition. For example, it can be mixed with water, saline, buffers, salts, stabilizers, preservatives, suspending agents and the like.

  Reporter gene expression can be measured while the mammal or cell is alive, or can be measured after solubilizing the cell. For example, to measure the expression of a luciferase gene in living tissue, Yamazaki, S. et al. (Science, 2000, 288) incorporated by reference as described herein with reference to photomultiplier tubes. 682-685), it is possible to measure bioluminescence continuously. The luciferase activity in solubilized tissue or cells can be measured using, for example, a Luciferase Reporter Assay System (Promega). Reporter gene expression can be measured temporally or spatially. Expression can also be analyzed by detecting the phase, amplitude, and / or period of the expression rhythm. The method of the present invention makes it possible to evaluate the immediate or long-term effects (including phase changes) of a compound. If these expressions are altered by administration of a compound, the compound becomes a drug candidate that regulates the expression of the Period 1 gene. Such compounds are expected to be applied as pharmaceuticals against various circadian rhythm disorders including sleep disorders. For example, an agent that resets or initiates a reporter gene expression oscillation would be expected to reverse or advance the pacemaker phase. Thus, these agents can be used to direct a desynchronized expression pattern to normal synchronization. The pharmaceutical product screened by the present invention is administered to the transgenic mammal of the present invention induced to become a circadian rhythm disorder model in order to evaluate the therapeutic effect of the drug.

When detecting gene expression in transgenic mammals, there are no particular restrictions on the organs to be measured, including the central nervous system (CNS), including the hypothalamic suprachiasmatic nucleus (SCN), and the peripheral nervous system (PNS) And other peripheral tissues including but not limited to liver, lung, and skeletal muscle. The system disclosed in the present invention is useful for assessing the phase relationship and synchronization mechanism of Period 1 expression in SCN and peripheral tissues. Here, when the weak light image obtained by the light emission microscope according to the present invention is further continuously or intermittently obtained at different desired elapsed times, it relates to one or more identical cells. Based on the analysis data obtained by comprehensively analyzing the light intensity for each time, for example, the activity pattern of the time gene and the response pattern of the intracellular substance due to the drug can be comprehensively evaluated. In addition, the cells that do not exhibit a predetermined light intensity or light distribution among the recognized cells are not subjected to exhaustive evaluation, so that accurate evaluation can be performed excluding cells that should not be analyzed. Further, by calculating the total value or the average value of the light intensities of all the cells subjected to the image analysis, it becomes possible to perform the evaluation of the analyzed whole cells in addition to the evaluation of the individual cells. Further, by classifying the cells into the same or different cell groups according to the light intensity and / or light intensity pattern of two or more cells subjected to the image analysis, the activity can be evaluated for each analyzed pattern. In some cases, it becomes possible to examine details of activity or activity change for each cell having a different pattern. According to the present invention, it is possible to perform analysis with various combinations of fluctuation parameters such as waveform shape ( amplitude length, period width, etc.) and waveform intensity ( expression amount, activation rate, etc.) of the expression pattern of a clock gene. it can. The results of the clock gene waveform analysis provide important information for research use such as diagnosis, treatment, growth (or biological development), and industrial (medicine, agricultural, etc.) applications. .

  The apparatus of the present invention can hold a biological sample containing a large number of cells in a state in which an image can be acquired, and can store weak optical data emitted from the biological sample and analyze the image. Weak light image acquisition means for acquiring image information, and image analysis means for comprehensively evaluating the light intensity of the weak light related to the recognized cells while morphologically analyzing the weak light image to recognize individual cells It is characterized by comprising. Here, since the holding means has a configuration in which a plate in which a plurality of wells are integrated is held so as to be addressable, evaluation between the plurality of wells is performed in the same field of view or in a predetermined order. It becomes possible to compare and correlate the results of activity evaluation with different samples or different drugs. In this case, the holding unit may be configured to hold a plurality of independent containers in an addressable manner, and is not limited to the field of view of the image acquisition unit, and can evaluate a large number of containers. In addition, by having a control unit that controls to perform evaluation according to the time of image acquisition, analysis of the same cell for each elapsed time, cells of different times (identical or different cells) exhibiting a specific activity Various time analysis such as comparative analysis can be performed. Further, by further including display means for displaying the image analysis result in association with image information, it is possible to display an image corresponding to the result desired to be viewed as an image from the analysis result. In addition, since the display means has a configuration in which desired image information is displayed as a moving image, a change in activity relating to one or more desired cells can be observed with a real-time image. As a moving image display, it is more preferable to enhance the sense of reality by superimposing weak light images of the same cell for each time by image processing. Further, a plurality of time-series images related to the same cell may be displayed in parallel (or just partially shifted) by frame advance so that the entire image for each time can be displayed.

  The system of the present invention can be used to identify a number of factors presumed to modulate Period 1 expression. Once new in vivo factors and genes related to circadian rhythm are identified using the system of the present invention, the in vivo oscillations of expression of these factors and genes can be assessed. Thereby, it is possible to isolate factors that control the vibration phase of SCN and peripheral tissues. These are considered to be novel genes and proteins involved in circadian rhythm, and by using these as targets, it is considered that screening for new drugs becomes possible. Such screening can be done both in vivo and in vitro.

Specifically, the in vivo screening method using the transgenic mammal of the present invention is achieved by the method (4) including the following steps.
Method (4): (a) administering a compound to a transgenic mammal whose circadian rhythm has already been determined; (b) periodically detecting the expression level of the reporter gene in the transgenic mammal, and expressing the rhythm (C) comparing the expression rhythm of the reporter gene after administration of the compound with that before administration; and (d) selecting a compound that alters the phase, period, or amplitude of the expression rhythm.

  The expression rhythm of the reporter gene can be determined by a method of detecting the expression rhythm of the reporter gene in the living animal body; a method of continuously observing expression fluctuations by culturing the excised tissue; or an extract of animal tissue It can be detected by a method that is prepared periodically and detects the expression level at each time point. For example, luciferin is administered to a transgenic animal at an appropriate timing by a suitable method (eg, intravenous injection, intraperitoneal administration, intraventricular administration, etc.). Subsequently, the animal is anesthetized, and the luciferase luminescence is measured by a CCD camera to determine the expression site and expression level of the reporter gene. This measurement is performed several times every few hours to confirm the expression rhythm of individual animals (Sweeney TJ et al., “Visualizing the kinetics of tumor-cell clearance in living animals ) ", PNAS, 1999, 96, 12044-12049; and Contag PR et al.," Bioluminescent indicators in living mammals ", Nature Medicine, 1998, 4, 245-247. ).

As described above, a novel drug can be screened in vitro using the present invention. Such an in vitro screening method can be achieved by the method (5) including the following steps.
Method (5): (a) culturing a tissue or cell derived from the transformant of the present invention or the transgenic mammal of the present invention; (b) compound of the transformant or tissue or cell over an appropriate period of time; (C) a step of periodically detecting the expression level of the reporter gene; and (d) the expression rhythm (phase, cycle, and amplitude) of the reporter gene after the treatment of (b). Selecting a compound to be modified.

  As used herein, the tissue or cell to be imaged of the present invention may be a primary culture or an established cell line cell. Although there is no restriction | limiting in the tissue, cell, etc. which are used by this specification, SCN in a vertebrate, a hypothalamic marginal cell, a peripheral nerve, etc. are preferable. The treatment with the compound may be performed, for example, by immersing tissues, cells, and the like for a certain period in a solvent to which the compound has been added. When measuring the change in the expression rhythm of the reporter gene, comparison may be performed using the same tissue or cell in which the expression rhythm has been determined in advance, or a control tissue or cell that has not been treated with the compound.

  In the in vivo and in vitro screening methods described above, stimulation treatment such as light stimulation may be performed together with administration or treatment of the compound.

  A compound identified by the test or screening method of the present invention can be used as a pharmaceutical agent for a desired circadian rhythm disease or disorder. These agents can be formulated as pharmaceutical compositions by appropriately combining with pharmaceutically acceptable carriers, solutes and solvents. The drug can be applied to diseases or disorders such as jet lag syndrome, sleep disorder due to shift work, sleep phase regression syndrome, and irregular sleep-wake disorder.

When the compound isolated by the screening method of the present invention is used as a pharmaceutical, it can be directly administered to a patient, or it can be formulated into a pharmaceutical composition by a known pharmaceutical formulation method. You can also For example, it can be administered after appropriately combining it with a pharmaceutically acceptable carrier or vehicle, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent and the like. The pharmaceutical composition of the present invention may be in the form of an aqueous solution, tablet, capsule, troche, buccal tablet, elixir, suspension, syrup, nasal solution, inhalation solution and the like. The content of the compound may be determined as appropriate. These can be administered to a patient, for example, usually by intraarterial injection, intravenous injection, subcutaneous injection, or oral administration, and such methods are known to those skilled in the art. The dose varies depending on the patient's weight, age, administration method, and symptoms, but those skilled in the art can appropriately select the dose. In general, the dosage varies depending on the effective blood concentration of the drug and the metabolic time, but the daily maintenance dose is about 0.001 mg / kg to 1 g / kg, preferably 0.01 mg / kg to 100 mg / kg, more preferably Is considered to be 0.1 mg / kg to 10 mg / kg. Administration may be from once to multiple times per day. If the compound can be encoded by DNA, the DNA can be incorporated into a gene therapy vector for gene therapy.

In addition, according to the present invention, the following software invention for an imaging apparatus is also included.
Software 1: In the apparatus according to any one of the apparatuses 1 to 17 described above, a program for causing the control unit and the image acquisition unit to function so as to execute interval imaging according to a set interval imaging condition. Software for high-throughput imaging devices.

It is a figure which shows an example of a structure of the apparatus for enforcing the luminescent sample imaging method concerning this invention. It is a figure which shows an example of the objective lens 2 which described the value of the square of (NA / (beta)). It is the figure which showed the objective lens simply. It is a figure which shows an example of the square of the brightness (NA / (beta)) of the image by the objective lens of the common microscope marketed now. It is a figure which shows the image which imaged the bright field image of the sample with the weak light imaging device shown in FIG. It is a figure which shows the image which exposed and imaged the image by the self-light-emission of the sample for 1 minute with the weak light imaging device shown in FIG. It is a figure which shows the image imaged by exposing the image by the self-light-emission of the sample for 5 minutes with the weak light imaging device shown in FIG. It is a figure which shows the image which accumulated and displayed the image shown to FIGS. 5-1 and FIGS. 5-3. It is a figure explaining the preparation method of the sample of the experiment by the weak light imaging device shown in FIG. It is a figure explaining the preparation method of the sample of the experiment by the weak light imaging device shown in FIG. It is a figure which shows the image which imaged the bright field image of the sample with the weak light imaging device shown in FIG. It is a figure which shows the image which exposed and imaged the image by the self-light-emission of the sample for 1 minute with the weak light imaging device shown in FIG. It is a figure which shows the image which overlapped and displayed the image shown to FIGS. 7-1 and FIG. It is a figure which shows the result of having measured the time-dependent change of the emitted light intensity from the sample corresponding to predetermined two area | regions among the images shown to FIGS. 7-3. It is a figure which shows a partial structure at the time of adding an epifluorescence apparatus to the weak light imaging device shown in FIG. It is a figure which shows a partial structure at the time of adding a filter unit to the feeble light imaging device shown in FIG. It is a figure which shows the light emission characteristic of a polychromatic luciferase gene, and the transmittance | permeability characteristic of the wavelength extraction filter shown in FIG. It is a figure which shows a partial structure at the time of adding a spectroscopic unit to the weak light imaging device shown in FIG. It is a figure which shows a partial structure at the time of adding a mirror switching bright field imaging unit to the feeble light imaging device shown in FIG. It is a figure which shows the structure in the case of arrange | positioning the weak light sample imaging device shown in FIG. 1 in a light-shielding device, and performing automatic control from the outside. It is a figure which shows the structure of the container which hold | maintains the sample 1 shown in FIG. 1 inside, and an environmental condition is variable. It is a figure which shows systematically each part structure applicable to the feeble light imaging device (or feeble light imaging unit) shown in FIG. FIG. 3 is a diagram showing conditions for the numerical aperture (NA) and projection magnification (β) of the objective lens used in Example 1. FIG. 6 is a diagram showing conditions for the numerical aperture (NA) and projection magnification (β) of the objective lens used in Example 2. It is a figure which shows the light emission image of the HeLa cell imaged in Example 2. FIG. FIG. 6 is a diagram illustrating a numerical aperture (NA) condition of an objective lens used in Example 3. It is a figure which shows the light emission image of the HeLa cell imaged in Example 3. FIG. It is the figure which showed the relationship between the square value of the (NA / (beta)) of the objective lens used in Example 3, and the light emission intensity of the light emission image shown in FIG. It is a conceptual diagram which shows the whole structure regarding the high-throughput imaging device as an application example of this invention. It is an internal block diagram of the imaging device of this invention which integrated the culture apparatus and microscope apparatus which can be applied to this invention. An electrically controllable unit in a high-throughput imaging apparatus is shown in a block diagram. It is a figure which shows the upper side figure (plan view) of the movable circular tray which hold | maintains a sample container in a high-throughput imaging device. It is a flowchart which shows the operation step in a high-throughput imaging device.

Explanation of symbols

1: Sample 2: Objective lens 3: Condensing lens 4: CCD camera 5: Monitor 101: Culture microscope main body 102: Controller 103: Warning buzzer 104: Warning display device 104a: Touch panel 105: Focus handle / joystick 106: Temperature controller 107 : Tank 108: Solenoid valve 109: Computer 110: Network 111: Remote computer 112: Heater 20: Incubator room 21: Totte 22: Lid 23: Hinge 24: Air piping 25: Sample container 26: Circular tray 27: Detachment of circular tray Sensor 28: Lid opening / closing sensor 29: Rubber 30: R stage motor 31: θ stage motor 32: Z stage motor 33: Objective lens 34: Rotation base 35: θ rotation axis 36: Linear movement base 8: Lead screw 39: Lead screw 40: Passing window 41: LED illumination 42: Fluorescent cube 43: Magnification changing lens 44: Mirror 45: CCD camera 46: Lens turret motor 47: Cube turret motor 48: Shaft 49: Shaft 50: Sheet 51 : Ring-shaped protrusion 52: Sample mounting hole 53: Nut 54: Linear guide 55: Base 56: Bellows 57: Sample container lid 58: Motor chamber 59: Microscope chamber 60; External control system 70; Interval imaging condition setting means

Claims (4)

A sample holder that maintains a living sample in a living and light-shielded environment, including at least cells that generate faint light;
Image acquisition means for imaging the biological sample present in a plurality of imaging regions and acquiring a sample image;
Control means for controlling the image acquisition means, and performing multi-point time lapse interval shooting for each imaging region,
The control means performs the resetting of the interval time or the imaging time in the multi-point time-lapse interval imaging based on the imaging time required for acquiring the sample image and the number of the imaging regions, thereby A biological sample detection apparatus comprising interval imaging condition setting means for setting conditions for interval imaging for each. The apparatus of claim 1.
A moving means for moving the image acquiring means relative to the plurality of imaging regions ;
The interval time is the total of the moving time, shooting time and control time for shooting the first time when performing multipoint time lapse, and the waiting time until immediately before starting the second shooting when performing multipoint time lapse. biological sample detection apparatus according to claim der Rukoto those. The apparatus of claim 1.
The image acquisition means has an imaging magnification changing means capable of changing an imaging magnification at the time of imaging,
A biological sample detection apparatus, wherein an interval imaging condition is set according to an imaging magnification by the imaging magnification changing means. The apparatus according to any one of claims 1 to 3, further comprising a culture means for continuously culturing cells in the biological sample, wherein the control means relates to each culture period relating to cells present in a plurality of imaging regions. A biological sample detection apparatus, characterized in that interval imaging conditions are set according to the imaging time during.