JP4800801B2 - Closed reaction detector - Google Patents

Description

  The present invention is a closure for detecting a biological activity in a test sample by reacting a biological activity in a biological sample such as a cell or tissue with a predetermined reagent or the like so as not to impair the activity as much as possible. The present invention relates to a mold reaction detector. The present invention also provides a reaction detection apparatus that can easily perform a necessary operation on a test sample at any time while maintaining constant culture conditions such as cells and biological tissues and / or light-shielding conditions for a long time.

  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 into which the luciferase gene has been introduced 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 to be 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. In order to sufficiently secure the measurement sensitivity by the luminometer, a general-purpose substrate solution such as firefly luciferin was treated at a concentration of 1 mM or more. In this concentration setting, since the cells are taken out and processed, they are not processed in a living state that can be returned to the culture environment. 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. Regardless of the culture period, the reagent conditions for luminescence are not changed.

In the above description, for example, a method and an apparatus for analyzing gene expression using a fluorescent protein as a reporter gene are disclosed in Patent Document 1 (Japanese Translation of PCT International Publication No. 2004-5000576). A method and apparatus for analyzing gene expression by bioluminescence using a luminometer is disclosed in Patent Document 2 (Japanese Patent Laid-Open No. 2005-1118050).

Special table 2004-500576 JP 2005-1118050 A

  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. In an apparatus that performs detection using such weak light, it is necessary to dispense a substrate solution or stimulate a drug or the like while maintaining a state where it is shielded from external light as much as possible. In addition, in a test sample containing living cells, it is necessary to perform various reactions and light detection while maintaining predetermined culture conditions.

  The present invention has been made in view of the above problems, and reacts with a predetermined reagent or the like so that the biological activity in a biological sample such as a cell or tissue is not impaired as much as possible. An object of the present invention is to provide a closed reaction detection device for detecting biological activity in a sample. In addition, the present invention provides a reaction detection apparatus that can easily perform necessary operations on a test sample at any time while maintaining constant culture conditions such as cells and biological tissues and / or light-shielding conditions for a long time. This is a further purpose.

The closed environment type reaction detection apparatus of the present invention includes a housing for maintaining a test sample in a closed environment closed from the outside, detection means disposed inside the housing, and a sealed state inside the housing. And an operating means for performing necessary operations on the test sample, and a remote driving means for driving the operating means manually or automatically from the outside of the housing.
Here, the detection means includes optical imaging means for performing optical imaging, so that weak light can be detected. In addition, when the housing includes a light shielding member for optically closing the outside, it is possible to detect weak light at a high S / N ratio. The housing is suitable for maintaining the biological activity of the test sample by including environmental condition adjusting means for adjusting the physical environmental condition and / or chemical environmental condition inside the housing. It becomes. In addition, since the environmental condition adjusting means includes a culture means for keeping living cells alive, reaction detection can be performed while maintaining biological activity constant for a long period of time. Further, when the operation means is a liquid supply means for supplying a reagent or the like in an adjustable manner, various reactions (e.g., luminescence reaction, stimulation reaction, etc.) on the test sample can be performed without any trouble in a closed environment.

  The liquid supply means includes a nozzle positioned in the vicinity of the test sample, a liquid feeding tube connected to the nozzle at the downstream end and extending in a sealed state outside the housing; When connected to the syringe mechanism for handling the liquid in the nozzle at the upstream end of the liquid supply tube, the timing and amount of liquid supply of the reagent etc. to the test sample from the outside of the housing It can be controlled properly. Further, when the syringe mechanism has a manual operation unit for discharging a necessary liquid from the nozzle, the user can supply the liquid at a desired timing as needed. Further, when the liquid feeding tube is flexible, the necessary liquid supply can be performed even if the manual operation unit is arranged at a desired position. Further, when the operation means is a physical stimulation means for executing physical stimulation on the test sample, a necessary stimulus response to the test sample is performed at a desired timing while maintaining a closed environment. It is possible. The housing has a double structure of an outer housing and an inner housing. The test sample is accommodated in the inner housing, and the remote driving means is disposed outside the inner housing and inside the outer housing. When the remote housing is disposed, the required operation can be performed by opening the outer housing in an open state when remote driving is desired while maintaining the closed environment in the inner housing. Further, the housing has a double structure of an outer housing and an inner housing, and the test sample is accommodated in the inner housing, and the optical imaging means is disposed outside the inner housing and inside the outer housing. In this case, stable optical imaging can be performed without being affected by a closed environment. Here, the optical imaging means may be accommodated in a separate housing from the inner housing under light-shielding conditions.

  Further, a housing for maintaining the test sample in a closed environment closed from the outside, a detection means for detecting a light signal generated from the test sample, and the inside and outside of the housing via a liquid feeding tube A nozzle is arranged to maintain a sealed state, and a nozzle for supplying a necessary reagent or the like to the test sample is disposed at the downstream end of the liquid feeding tube, and the liquid is fed to the upstream end of the liquid feeding tube. When equipped with a liquid feed control unit for controlling the amount of liquid or gas that moves as a liquid transfer medium in the tube, accurate liquid supply operations can be performed outside the housing while maintaining a closed environment. There is an advantage of being able to. In addition, when at least a part of the housing is formed integrally with the opening / closing cover and a syringe mechanism for manually or automatically driving the dispensing means is disposed outside the opening / closing cover, the opening / closing cover is opened. Thus, the liquid supply to the test sample can be easily performed, and the closed environment can be easily reproduced by closing the closed cover. In particular, according to the present invention, since the dispensing means has a catheter structure, accurate liquid supply can be performed from the outside of the housing to the test sample in the housing.

  According to the applicant's investigation, it was also found that by changing the substrate concentration setting according to the culture period, it is possible to simultaneously obtain a luminescent image and maintain the biological activity of the cell for a long time. It was issued. Therefore, it is preferable that the reaction detection apparatus of the present invention can be configured so that the dispensing operation of the substrate solution by the liquid supply means can be selected according to the concentration because detection corresponding to various culture periods can be executed. As a substrate concentration to be specifically selected, for example, when the culture period is several days or more, the substrate is preferably set to a concentration of 700 μM or less. When the culture period is less than several days, the substrate is preferably at a concentration of 800 μm or more and 1 mM or less. By setting the substrate concentration to 200 μM or more, it becomes possible to obtain a stable luminescence reaction amount in any culture period. Examples of the substrate contained in the substrate solution include firefly luciferin or coelenterazine. Even in the apparatus of the present invention, it is possible to keep the amount of luminescence that can be imaged constantly during the culture period while maintaining the state where the biological activity is not lowered by continuing the culture in the substrate concentration range. preferable. Based on the luminescence image of the cells, the living cells are treated with the luminescent component and the substrate solution for inducing luminescence to the luminescent component is present in an appropriate culture environment to cause the cells to emit light. In performing the analysis, while collecting the luminescence from the cells under the optical condition that the square value of (NA ÷ β) expressed by the numerical aperture (NA) and the projection magnification (β) is 0.01 or more, By setting the substrate concentration to 600 μM or less, both optical imaging and luminescence reaction can be performed in a good state, which is suitable for a live cell image analysis method. Furthermore, when the square value of (NA ÷ β) as the optical condition is 0.039 or more, the result of optical imaging can be detected with high sensitivity in a short time. It is possible to obtain a luminescent image with an imaging time of 30 minutes or less while the substrate concentration is 200 μM or more. In the present invention, in order to optically image a luminescent sample, an objective lens having a numerical aperture (NA) and a projection magnification (β) (NA ÷ β) 2 value of 0.01 or more is used. Thus, there is an advantage that an image of only light emission can be imaged in a short time. If a luminescent image can be obtained in a short time, it is possible to guarantee a sufficient image quality for accurate image analysis. In addition, according to the same optical conditions, there is an advantage that small and economical imaging can be performed without using an expensive cryogenic cooling type imaging device.

  In particular, by using an objective lens with a numerical value (NA) and a projection magnification (β) of (NA ÷ β) 2 of 0.039 or more, imaging can be performed at a significantly higher speed than before. This is preferable in that image analysis of easily movable cells (neuronal cells, cyanobacteria, etc.) can be reliably performed.

  Furthermore, by using the objective lens in a short time of 1 to 5 minutes when the value of (NA ÷ β) 2 expressed by the numerical aperture (NA) and the projection magnification (β) is 0.071 or more, This is preferable because real-time imaging can be performed in any cell analysis.

  Further, the present invention makes it possible to perform image analysis at short time intervals using an objective lens having a high numerical aperture (NA) for luminescence imaging, so that all stimulus responsiveness is not missed. This provides an excellent method in drug discovery and diagnosis. In addition, a luminescent sample (eg, a luminescent protein (eg, a luminescent protein expressed from an introduced gene (eg, luciferase gene)), a luminescent cell or a collection of luminescent cells, Even a tissue sample or a luminescent individual (such as an animal or an organ) can be analyzed with a clear exposure time and in real time.

According to the closed-type reaction detection apparatus of the present invention, the biological activity in a biological sample such as a cell or tissue is reacted with a predetermined reagent or the like so as not to impair the activity as much as possible. Activity can be detected. In addition, necessary operations on the test sample can be performed easily and at any time while maintaining constant culture conditions such as cells and biological tissues and / or light-shielding conditions for a long time.

  Hereinafter, embodiments of a closed reaction detection device and a faint light analysis method using the device will be described in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  A configuration of an apparatus for carrying out the method according to the present invention will be described with reference to FIG. FIG. 1 is a schematic diagram showing the configuration of the first embodiment of the reaction detection apparatus of the present invention. As shown in FIG. 1, a sample 1 that is an imaging target arranged at a predetermined position is to be imaged with a short exposure time, and in real time, an objective lens 2, a condensing lens 3, a CCD camera 4, and a monitor. The attached computer 5 and the instruction input means 7 detect the luminescence reaction based on a predetermined instruction. In addition, a dispensing means 8 for dispensing a luminescent reagent (for example, a substrate solution) or a stimulus reaction reagent (for example, an anticancer agent) to the sample 1 is provided with a reagent container 10 and a sample by a transfer drive unit 9. It is configured to be transferred three-dimensionally (XYZ) between 1. The computer 5 controls the imaging operation by the CCD camera 4 and the dispensing operation of various reagents by the dispensing means 8 according to a predetermined reaction schedule. The apparatus may further include a zoom lens 6 as shown. Here, all of the above-described components except for the computer 5 and the instruction input means 7 are stored in a sufficiently light-shielded environment by a housing 11 designed to ensure light-shielding properties.

  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, luminescent individuals (animals, etc.), etc., in which a predetermined container (eg, culture dish, microplate, etc.) uniformly contains a substrate having an effective concentration in advance. Live cells are placed on the medium for use. Here, the case of the luminescent cell which introduce | transduced the luciferase gene as the sample 1 is demonstrated. The objective lens 2 has a numerical value (NA ÷ β) 2 expressed by a numerical aperture (NA) and a projection magnification (β) of 0.01 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 of the computer 5 outputs an image captured by the CCD camera 4. If necessary, display the luminescence image of the cells after optical imaging on the monitor as shown in the figure, and apply the necessary stimulating reagent to the specific cells (arrows in the figure) to be dispensed. Dispensing is performed at the specified timing. This dispensing operation can also be observed by the monitor of the computer 5.

  The value of (NA / β) 2 is written in the objective lens 2 and the packaging container (package) of the objective lens 2. The conventional objective lens indicates the lens type (eg “PlanApo”), magnification / NA oil immersion (eg “100 × / 1.40 oil”) and infinity / cover glass thickness (eg “∞ / 0.17”). It had been. However, the objective lens (objective lens 2) of the imaging means according to the method of the present invention includes a lens type (for example, “PlanApo”), magnification / NA oil immersion (for example, “100 × / 1.40 oil”), infinity / In addition to the cover glass thickness (for example, “∞ / 0.17”), an injection opening angle (for example, “(NA / β) 2: 0.05”) is also described.

  FIG. 2 is a schematic diagram showing the configuration of the second embodiment of the reaction detection apparatus of the present invention. In FIG. 2, compared to FIG. 1, a so-called inverted type imaging system in which the set of the objective lens 2, the condenser lens 3 and the CCD camera 4 constituting the optical imaging means is arranged below the sample 1. Is different. This set of optical imaging means is arranged outside the housing 11. By doing so, the influence on the optical element such as the objective lens 2 can be avoided even if the inside of the housing 11 is maintained for a long time under a high humidity condition of 37 ° C. advantageous for culture. However, by adjusting the temperature of the sample 1 and the objective lens 2 so as to be almost the same temperature by an appropriate temperature control means 12, the objective lens 2 during imaging can be prevented from being clouded or condensed, and stable for a long time. The luminescent image can be acquired.

  FIG. 3 is a schematic view showing the configuration of the third embodiment of the reaction detection apparatus of the present invention, and shows a modification obtained by partially improving the second embodiment. That is, in FIG. 3, compared with FIG. 2, the dispensing means 8 is fixed at the center of the accommodating portion for accommodating the sample 1, and instead, the mounting table 13 for holding the sample 1 is provided with the transport driving means. 14 can move freely in the XY directions. This moving range is preferably such that the sample 1 can be positioned without fullness with respect to the dispensing means 8 at the fixed position. The dispensing means 8 repeatedly dispenses a reagent from a predetermined reagent container 10 at a timing instructed in advance via an automatically controlled syringe mechanism 15. In this way, it is possible to perform a desired order and number of luminescent reactions or stimulation reactions on a plurality of sample components (for example, cells) in the sample 1. The substrate solution may be set to be added after a predetermined time so as to keep the luminescence reaction constant. In this case, by cooperating with the operation of moving the sample 1 evenly, The operation | movement which stirs the inside substrate solution uniformly is also possible.

    FIG. 3 further shows a housing 11 (referred to as a first housing) for maintaining the sample 1 and the dispensing means 8 in a predetermined reaction or culture environment condition, and a main body side housing that appropriately blocks outside air and extraneous light from the entire apparatus. 16 (referred to as a second housing). The first housing 11 can be opened and closed by manually holding the handle 17 by a shielding cover 17 that can be opened and closed upward, and the front cover 19 that can be opened and closed in the horizontal direction is manually installed on the upper portion of the second housing 16. It can be opened and closed by grasping with. As described above, since the double housings 11 and 16 are each provided with the shielding covers 17 and 19 that can be opened and closed, the user can secure a sufficient working space on the sample 1 while keeping extra light and / or outside air. Intrusion can be minimized. In the third embodiment, the covers 17 and 19 of the housings 11 and 16 are laid out so that the opening and closing directions are the vertical and horizontal directions so that the same working space is a minimum opening and closing space. This has contributed to maintaining a good closed environment for the sample 1.

  FIG. 4 is a schematic diagram showing the configuration of the third embodiment of the reaction detection apparatus of the present invention. A computer 5 with a monitor is installed on the lower front portion of the shielding housing 11 constituting the main body so that only the monitor portion is exposed, and below that, the instruction input means 7 is installed so that only the operation keys are exposed. Has been. A petri dish container 21 in which a large number of cells are placed on a predetermined medium is placed in the center of the shielding housing 11, and the reagent injection nozzle 22 is fixed at a lower position inside the container. The reagent injection nozzle 22 is connected to a manual syringe 24 as a small driving means via a flexible tube 23 (for example, made of silicon) (FIG. 4B). The manual syringe 24 is designed to discharge a small amount of liquid quantitatively at a time (or divided) with appropriate dispensing accuracy. Here, the manual syringe 24 is usually slidably accommodated inside the upper housing 11 that also functions as an open / close cover. Even if the upper housing 11 that is also an open / close cover is opened and closed, the flexible tube 23 bends flexibly. Further, during this opening / closing operation, the manual syringe 24 is in a stably stored state, so that it does not operate by mistake. In the closed state of the housing 11, the user pulls out the manual syringe 24 from the housing 11 (FIG. 4C), and the manual syringe 24 is filled with a desired luminescent reagent previously filled in the flexible tube 23 by suction. An appropriate amount is injected into the petri dish container 21 by the discharging operation. When the injection is finished, the manual syringe 24 is pushed in the opposite direction to the drawn state and returned to the original stored state. On the other hand, since the above-described set of light emission imaging means is built in the housing 11 below the petri dish container 21, it is possible to continuously obtain light emission images in an appropriate constant temperature environment and a light shielding environment. In the fourth embodiment, a configuration in which the luminescence reaction can be continuously performed in a closed environment has been described. However, a plurality of manual syringes 24 are similarly installed so that stimulation reagents can be injected at a desired timing. It is preferable to do this. Further, without installing a plurality of manual syringes 24, it is also possible to sequentially suck a plurality of different reagents into the same flexible tube so that a plurality of different reagents can be discharged in order. In particular, the manual syringe 24 can suitably utilize a commercially available catheter for liquid injection. By using a catheter, there is an advantage that a small-sized dispensing means having a suitable dispensing accuracy and good flexibility can be installed. The catheter is also preferable in that it has a long and narrow nozzle shape at the tip, and can be disposed with high accuracy at an appropriate portion of the test sample in the container. Of the overall operation, the automatic control operation set by the instruction input means 7 is started and stopped by the on / off operation of the operation changeover switch 25.

  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 (NA ÷ β) 2 represented by the numerical aperture (NA) and the projection magnification (β). The value is 0.01 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. The objective lens 2 is expressed by the numerical aperture (NA) and the projection magnification (β) (NA ÷ β) 2 on the objective lens 2 and / or a packaging container (package) that wraps the objective lens 2. Value (for example, 0.01 or more) was expressed. Thus, for example, a person who observes a luminescent image can easily find an objective lens suitable for imaging a luminescent sample with a short exposure time and in real time if the value of (NA ÷ β) 2 is confirmed. You can 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, (NA / β) 2. Here, the objective lens generally has a relationship of the following Equation 3 between the incident aperture angle NA and the exit aperture angle NA ′, and NA ′ 2 indicates the brightness that reaches the observer's eyes, a CCD camera, or the like. Value.
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 value of image brightness (NA / β) 2 in a general microscope objective lens currently on the market was examined, it was in the range of 0.0005 to 0.002.

However, using a microscope equipped with a commercially available objective lens as described above, for example, even if a cell expressing a luciferase gene and illuminating it is observed, the light emitted 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 a large NA and a small β as compared with the above-described objective lenses generally on the market. Therefore, NA′2 of the objective lens of the present invention is a large value. That is, it can be said that the objective lens of the present invention is a bright objective lens. 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. Moreover, the objective lens of the present invention has a large diameter of several to about 10 cm. Thereby, a moving luminescent sample that 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.

  The measurement principle for implementing the method of the present invention, the configuration of the optical system, and the like have already been described with reference to FIG. 1, but refer to the applications by the present applicant (Japanese Patent Application Nos. 2005-267531 and 2005-44737). May be. As described in the above-mentioned application, the applicant, through optical experiment, has a square value of (NA ÷ β) represented by a numerical aperture (NA) of the objective lens and a projection magnification (β) of 0. By taking an image with an optical system of 0.01 or more, proof data that can be imaged only by light emission generated from a single cell was obtained. Furthermore, the present applicant has proceeded with studies and found that the variation pattern of gene expression is different among a plurality of cells cultured in the same petri dish. Surprisingly, the above imaging conditions can also be applied to weak luminescent images handled in the application examples implemented in the present invention, and weak such as bioluminescence in a short time (for example, 1 to 20 minutes). Cell images can be captured with various luminescent components. Further, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) is 0.071 or more, the objective lens of the imaging device can be imaged within 1 to 5 minutes. It was also found that cell images that can be analyzed can be provided.

  As shown in FIG. 1 described above, the apparatus for carrying out the 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, and has been conventionally employed. A high numerical aperture (NA) objective lens 2, a CCD camera 4 and a monitor 5 are basically configured. In the present invention, an apparatus having these basic configurations is referred to as a light emission microscope. In order to perform imaging in a dark field, the light emission microscope is preferably accommodated by a light shielding lid or housing as appropriate. Moreover, imaging can be automatically performed over a long period of time by combining a culture apparatus capable of maintaining appropriate culture conditions integrally with a light emission microscope. In addition, if it is a structure which has an imaging mechanism, it does not need to be in the form of a microscope, and may be in the form of a measuring instrument such as a microplate reader.

Next, the configuration, form, and operation of the light emission microscope used in the present invention will be described. The basic feature of the luminescence microscope is that light emitted from a photoprotein expressed in cells in the microscope field can be monitored in a short time by imaging with a digital camera through an objective lens (NA 0.75) and an optical filter. That is. Since a gene expression pattern can be obtained in real time as a luminescence image, it is a technology that is expected to be applicable to a wide range of research subjects in gene expression assay systems using cells. The basic measurement system consists of a sample observation unit that is an optical system adjacent to the culture apparatus unit, and the light emitted from the optical system is captured by a digital camera through an objective lens (NA 0.75, preferably NA 0.75 or more) and an optical filter. Later, data recording and analysis are performed on a digital image capturing personal computer. The culture apparatus part can be kept warm and moisturized by a heat plate and a chamber. The temperature in the culture apparatus is set to 25-37 ° C and the sample is observed. The sample is preferably observed at 37 ° C. The humidity is set to 0 to 100% and observed. Preferably, it is set to 60 to 100%. Furthermore, by acquiring a bright-field image in the same field as when optical luminescence imaging was performed, and superimposing the light-emission image and the bright-field image with image analysis software or the like, it is possible to identify the cells that emit light. .

Definitions of terms relating to the method of the present invention are shown below.
<Promoter region of a gene whose expression is induced by stimulation>
Examples of the gene promoter region whose expression is induced by stimulation in the present invention include a promoter of an early gene. Examples of the promoter of the early gene used in the present invention include the c-fos promoter region. In addition, the promoter used in the present invention includes a counterpart of any animal species of the promoter. Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 2000 bases upstream from the transcription site of the gene can be used.

<Reporter gene>
The reporter gene in the present invention means a gene encoding a reporter protein that emits detectable fluorescence. For example, luciferase derived from firefly or Renilla can be mentioned. Furthermore, the gene which codes (beta) galactosidase and the gene which codes alkaline phosphatase can be mentioned, for example. As a substrate for causing the reporter gene to emit light, any substrate can be applied, and in particular, firefly luciferin and oxyluciferin can be preferably applied.

<Optical imaging>
Optical imaging in the present invention means an imaging method for monitoring, recording and analyzing the presence, absence or intensity of a detectable signal emitted from a biological sample such as a cell or tissue. For example, to achieve optical imaging with a reporter gene in a cell into which the reporter gene has been introduced, the intensity of the signal emitted by the reporter gene is sufficiently sensitive so that the signal can be analyzed from outside the cell. There must be. Since optical imaging is readily applicable to automation, it can be used to monitor multiple gene expressions simultaneously. The technique for processing image information two-dimensionally or three-dimensionally at an arbitrary position of an imaged sample image is disclosed in Japanese Patent Application Nos. 2004-172156, 2004-178254, and 2004-342940 by the present applicant. Japanese Patent Application No. 2005-267531 may be referred to. The difference between time-series image acquisition in fluorescence observation and light emission observation is that there is no influence of extra light because light emission observation does not require optical scanning (laser scan) with excitation light. According to the imaging technique performed in the present invention, a luminescent image can be captured in real time.

B. Optical imaging method of gene expression <Production of gene expression vector>
When using animal cells, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of interest, and a stop codon. Also includes DNA encoding signal peptide, enhancer sequence, 5 'and 3' untranslated regions of gene encoding the protein, splicing junction, poly A addition signal, selectable marker region or replicable unit, etc. You may go out.

<Gene transfer method into cells>
Examples of methods for introducing genes into cells include calcium chloride method or calcium chloride / rubidium chloride method, lipofection method, electroporation method and the like. The gene-introduced cells used in the present invention include both transiently expressing cells and stable expressing cells.

<Optical imaging of gene expression by stimulation>
According to the present invention, it is possible to analyze the amount of luminescence in time series by a stimulus that contacts a cell with a substance capable of inducing any luminescent reaction. The cells to be stimulated and analyzed may be single cells or cell groups. By performing luminescence imaging, it becomes possible to trace any particular cell until what time, and the luminescence intensity or luminescence behavior due to the difference in cells can be accurately evaluated.

Use a device (for example, petri dish, multi-plate having many wells, etc.) capable of culturing a desired cell for the constant of the introduced cells (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ^{3 to} 1 × 10 ⁶ cells) And cultured in a desired nutrient medium (eg, D-MEM medium). A sample of this constant number of cells is preliminarily kept at a temperature optimum for the cells (for example, 25 to 37 ° C., preferably 35 to 37 ° C.) and water is injected to prevent the sample from being dried. A luminescent image is recorded by a digital camera through an objective lens in a sample observation section of the luminescence microscope that is installed in the culture apparatus. A substance (for example, a compound) for stimulating by contacting cells is added to the sample at a desired concentration (for example, 1 pM to 1 M, preferably 100 nM to 1 mM), and a desired time interval (for example, 5 minutes) is added. An image capable of image analysis can be prepared by recording a luminescent image in 5 hours, preferably 10 minutes to 1 hour. The image analysis of the prepared image can be automatically analyzed by analysis software that is commercially available or designed by a unique analysis algorithm. By displaying time-series parallel display or moving image on the display screen, analysis with the naked eye is also possible. In the embodiment described above, an operation such as reagent dispensing can be performed while displaying a light emission image on a monitor. Therefore, an appropriate operation such as appropriate dispensing can be easily performed while confirming an image even during long-term imaging. In the time-series moving image display, the presence or absence of an intermediate operation such as dispensing or the operation image may be displayed on the monitor at the same time for tracking management.

  In addition, this invention is not limited to description described in embodiment mentioned above, an Example, etc., A various aspect and its equivalent can be included. Further, the various processing conditions described in the present invention may be arbitrarily combined. Furthermore, it may be in the form of a product that is manufactured by performing the treatment method of the present invention and is stabilized so as to be distributed (for example, drying treatment, vacuum packaging, sheet shaping treatment, thickening saccharide solution impregnation treatment, etc.). . In addition, in the case of a two-dimensional shape cell group in which a living tissue is thinned, a bottom surface of a container such as a single chip substrate or a single petri dish may be covered with a necessary surface area, or many different living tissues Each of the two-dimensional shape cell groups obtained by subdividing can be microarrayed by spotting in a matrix at different address positions.

It is a figure which shows the outline | summary of the closed type reaction detection apparatus which is the 1st Embodiment of this invention. It is a figure which shows the outline | summary of the closed type reaction detection apparatus which is the 2nd Embodiment of this invention. It is a figure which shows the outline | summary of the closed type reaction detection apparatus which is the 3rd Embodiment of this invention. It is a figure which shows the outline | summary of the closed type reaction detection apparatus which is the 4th Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample 2 Objective lens 3 Condensing lens 4 CCD camera 5 Monitor 6 Zoom lens 7 Instruction input means 8 Dispensing means 9 Movement drive part 10 Reagent container 11, 16 Housing 12 Temperature control means 13 Sample mounting stage 14 Sample conveyance drive means 15 Syringe mechanism 17, 19 Opening / closing cover 18, 20 Handle 21 Petri dish 22 Injection nozzle 23 Flexible tube 24 Manual syringe 25 Operation switch

Claims (10)

A housing for maintaining the test sample in a closed environment closed from the outside, a detection means arranged inside the housing, and a necessary operation for the test sample arranged inside the housing in a sealed state A closed-environment type reaction detection device comprising: operating means for performing the operation; and remote drive means for manually driving the operating means from outside the housing,
The housing has a double structure of an inner housing in which the test sample is accommodated and an outer housing having a light shielding member for optically closing the specimen, and the housing Comprising environmental condition adjusting means for adjusting internal physical and / or chemical environmental conditions;
The environmental condition adjusting means comprises a culture means for maintaining viable cells alive,
The remote drive means is disposed outside the outer housing;
The detection means includes optical imaging means for performing optical imaging, arranged outside the inner housing and inside the outer housing,
A reaction detection apparatus, wherein the operation means is driven by the remote drive means while a user confirms an image detected by the optical imaging means on a monitor . The reaction detection apparatus according to claim 1 , wherein the operation unit is a liquid supply unit that supplies a reagent or the like in an adjustable manner. The liquid supply means, the nozzle positioned in the vicinity of the test sample, the liquid supply tube that is upstream extending kept sealed to the outside of the outer housing as well as connected at the nozzle and the downstream end The reaction detection device according to claim 2 , further comprising a syringe mechanism that is a remote drive unit for handling the liquid in the nozzle at an upstream end portion of the liquid feeding tube. The liquid supply tube is flexible der is, and, the reaction detecting device according to claim 3, wherein the syringe mechanism characterized that you have been able to store inside the housing the drawer. The reaction detection apparatus according to claim 1 , wherein the operation unit is a physical stimulation unit that performs physical stimulation on a test sample.   The reaction detection apparatus according to any one of claims 1 to 5, wherein the optical imaging means is an imaging means for observing the cells as a luminescent sample over time. A housing for maintaining the test sample in a closed environment closed from the outside;
Detection means for detecting a light signal generated from the test sample;
Dispensing means for supplying a necessary reagent to the test sample , disposed inside the housing ;
A liquid feed control unit disposed outside the housing ;
The housing comprises a culture means for maintaining the test sample alive,
The dispensing means comprises a liquid supply tube for connecting with the inside and outside of the housing to maintain a sealed state, and a nozzle connected to the downstream end of said transmission fluid tube,
The liquid supply tube is connected with Oite the liquid feed control section at its upstream end,
By the liquid feed control section, the amount of movement of the liquid to gas as a fluid transfer medium in the liquid supply tube is controlled,
A closed environment type reaction detection apparatus , wherein a user manually drives the dispensing means by the liquid feeding control unit while checking a detection result by the detection means on a monitor . At least partially integrally formed with the cover, the reaction detecting device according to claim 7, characterized in that the syringe mechanism is disposed of as the liquid feed control section to the outside of the cover of the housing. The reaction detection device according to claim 7 , wherein the dispensing unit has a catheter structure.   The reaction detection apparatus according to any one of claims 7 to 9, wherein the dispensing unit is a unit that dispenses a substrate solution for performing a luminescence reaction using the test sample as a luminescent sample.

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