JP4823632B2 - Method and apparatus for acquiring an image of a biological test sample using optical imaging means - Google Patents

Description

  The present invention relates to a method and apparatus for acquiring a signal from a test sample derived from an organism that emits faint light, and relates to a method and apparatus for observing or measuring using the acquired signal.

  In recent years, techniques for imaging biological samples such as cells using fluorescence and luminescence have played an important role in research in the life science field. Various biological phenomena occurring inside and outside cells can be actually analyzed by marking specific proteins and using luminescence, and their dynamic behavior can be obtained in real time. In particular, the use of fluorescent proteins such as GFP (Green Fluorescent Protein) has enabled stable and easy imaging of intracellular structures, and the analysis of life phenomena is an image analysis. Steady progress is being made through technology.

    In spectroscopic measurement, genes such as luciferase and aequorin, which are bioluminescent proteins, are often used as a reporter molecule for protein expression, and are used for analysis of intracellular functions. When performing various functional analyzes in the cell, the focus position of the lens is adjusted to a specific part of the cell, and a clear image is drawn, or a light signal from a specific protein is received efficiently. Has become very important.

  However, the light intensity of self-emission from living organisms is generally very weak and usually cannot be directly observed with the naked eye. In addition, particularly when a living sample such as a cell is to be observed for a long period of time, the cell may move during this observation period. In addition, the emission intensity may change during the observation period, and it is extremely difficult to identify which cells are emitting how and which cells are emitting light. This makes it very difficult to analyze changes in the cytoplasm and neurite emission in one cell. Accordingly, the present invention provides a method and apparatus for solving the conventional problems as described above, specifying a desired cell and a light emitting site in the desired cell, and acquiring data from the specified region. With the goal.

  The present applicant has first worked on imaging technologies related to various light emission phenomena and has been intensively studied (see Japanese Patent Application No. 2005-267531). According to this study, a single value is obtained by imaging with an optical system having a square value of (NA ÷ β) expressed by the numerical aperture (NA) of the objective lens and the projection magnification (β) of 0.01 or more. It was proved that it can be imaged only by luminescence generated from cells. Surprisingly, this imaging condition has the potential to be applied to any biological sample that has traditionally been difficult to image, such as bioluminescence in the short period of time (eg within 20 minutes) (and It is possible to take a cell image with a weak luminescent component that cannot be directly observed under a microscope). 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 in a short time of 1 to 5 minutes and that can be analyzed can be provided.

  Here, when observing the luminescence phenomenon due to bioluminescent protein, it is extremely weak luminescence and it is unclear which cell emits light, so the surrounding background light is blocked and usually a low magnification objective lens is used. The emission signal had to be acquired. In particular, the desired light-emitting site is often a part of the observation field, stays in a very small area, and the light intensity is weak, making it difficult to confirm its analysis and function. In order to magnify and observe a desired site in the observation field of view, it is usually sufficient to increase the magnification of the objective lens, but when acquiring a weak luminescence image such as a bioluminescent protein, the field of view becomes dark, There was also a problem that the luminescence image could not be easily confirmed.

Therefore, the present invention is a method for acquiring an image of a biological test sample using an optical imaging means, the step of illuminating the test sample and acquiring an illumination image, and illuminating the test sample. And a step of acquiring a luminescence image of a cell, a step of superimposing the illumination image and the luminescence image of the cell, and a step of designating an analysis region. Further, after the step of superimposing the illumination image and the light emission image of the cells, the superimposed image is displayed or recorded.

Furthermore, the present invention includes a step of acquiring a fluorescence image of a cell. Furthermore, the test sample is illuminated a plurality of times to obtain an illumination image. Furthermore, before or after the step of stimulating the cells, the step of illuminating the test sample to acquire an illumination image, and the step of acquiring the luminescence image of the cell without illuminating the test sample. It was decided that Further, the step of stimulating the cells is based on drug administration, electrical stimulation, gas, and heat. Further, the method includes a step of measuring the light intensity in the analysis region and a step of displaying a change in the light intensity. Furthermore, at least two of the illumination image of the designated area, the light emission image of the cell, and the fluorescence image of the cell are displayed.

The present invention also provides an apparatus for acquiring an image of a biological test sample using an optical imaging means, the means for illuminating the test sample, the means for acquiring the illumination image, and the light emission of the cells. And a means for acquiring an image, a means for superimposing the illumination image and the light emission image of the cell, and a means for designating an analysis region. Furthermore, it has means for displaying or recording the superimposed image. Furthermore, it has means to stimulate the cells. The means for stimulating the cells is at least one of a reagent supply means, a temperature adjustment means, and a gas supply means. Furthermore, it has the means to acquire the fluorescence image of a cell.

  According to the method and apparatus of the present invention, even if a cell moves during measurement, it is possible to specify which cell emits faint light (for example, luminescence) and to measure a change with time due to the faint light.

Hereinafter, the present invention will be described in detail with reference to the drawings.
1) The measurement procedure for carrying out the method of the present invention will be described with reference to FIG.
A test sample (cell) and a culture solution are put in a petri dish (sample container), and the sample is set in an apparatus.
Turn on lighting.
An illumination image of the test sample (cell) is acquired from the signal from the image sensor.
In the obtained illumination image, a desired area is designated, and an area having an appropriate cell density is designated.
Move the stage and move the appropriate cell density area specified to the center of the field of view.
The data of the illumination image is stored in the storage device.
Turn off the light.
Cells are stimulated using cell stimulation means. For example, the medicine is administered by an automatic dispensing device.
Illumination immediately after 8 may be performed, and illumination image data may be stored in a storage device.
A light emission signal is acquired without performing illumination.
The light emission signal is stored in the storage device.
Repeat steps 10 and 11 until a predetermined time, a predetermined light emission intensity, or a predetermined number of times is reached.
At this time, the acquisition of the illumination image and the acquisition of the luminescent image can be performed in a pair (when cells easily move), or the illumination image can be acquired every predetermined number of times. This pattern can be performed in a pattern more suitable for ease of cell movement, measurement period, and the like.
A superimposed image of the illumination image and the light emission image is created and displayed. By superimposing the illumination image and the luminescent image, it is possible to identify the luminescent cells. Therefore, even if the cells move during the measurement, the cells can be easily identified.
Judgment is made depending on whether or not the light emitting site of the cell can be identified. The measurement area can be specified by changing the threshold value by using a mouse or a cursor on the monitor or by performing image processing. For example, one cell can be used as the measurement region, and a part of the cells can be designated as the measurement region.
Judgment is made based on whether or not the light emitting site of the cell can be discriminated. If it is difficult to discriminate, the output from the CCD is displayed in a pseudo color to make it easy to see, and the measurement area is designated. For example, one cell can be used as the measurement region, and a part of the cells can be designated as the measurement region.
The change in emission intensity over time is displayed as an animation or displayed as a graph.
End.

Example 1
The configuration of the embodiment will be described with reference to FIGS. FIG. 2 is a schematic view of a weak light detection device based on an inverted microscope. The weak light detection device includes a light source 2, an illumination optical system 1 for guiding the light emitted from the light source 2 into parallel light, and guiding the light to the test sample, an observation optical system 5 for generating an image of the test sample 4, An eyepiece 6 for enlarging and visually observing an image of the test sample 4 and a CCD camera 8 having an image sensor 7 for capturing an image of the test object 4 are configured. A computer 16 also serving as a television monitor is connected to the CCD camera 8 by a signal cable.

  The illumination optical system 1 includes a collector lens 10, a deflection mirror 12 for deflecting the optical axis 11 of illumination light, and a condenser lens 14 in order from the light source 2 side. The light source 2 uses an incoherent light source having a visible wavelength such as a halogen lamp, an LED light source, a tungsten lamp, or a mercury lamp. Alternatively, the light of a coherent light source such as a laser may be changed to incoherent light using a diffusion plate or the like and used as a light source. Visible light is usually used as the wavelength of the light source, but infrared light may be used.

  The observation optical system 5 includes, in order from the test sample 4 side, an objective lens 15 for forming an image of the test sample 4, a first relay lens 16, a deflection mirror 17 for deflecting light from the objective lens 15, a first mirror. The first relay lens 16 and the second relay lens 18 for forming an image of the objective lens 15 (image of the test sample 4) on the imaging surface. Further, a switching mirror 20 is provided between the second relay lens 18 and the imaging plane 19 so that the visual observation with the eyepiece 6 and the observation with the CCD camera 8 can be arbitrarily switched with respect to the image of the test sample 4. Is arranged. In addition to the mechanical switching type, the switching mirror 20 may use a half mirror to separate the two optical paths.

  The light source 2 is collimated by the collector lens 10 and projected onto the pupil position of the condenser lens 14. The image of the light source 2 illuminates the test sample 4 as Koehler illumination by the condenser lens 14. The light that illuminates the test sample 4 passes through the test sample 4 and enters the objective lens 15. The measurement light incident on the objective lens 15 forms an image of the test sample 4 on the image plane 19 by the objective lens 15, the first relay lens 16 and the second relay lens 18. Here, an objective lens with a magnification of 20 is used. The image of the test sample 4 formed on the imaging surface 19 is incident on the eyepiece 6 as it is, and is imaged on the image sensor 7 of the CCD camera 8 by the switching mirror 20.

  The light receiver uses a CCD (Charge Coupled Device) camera. The number of pixels of the image sensor on the light receiving surface of the CCD camera 8 is 1,360 × 1,024, for example. Since the light emitted from the test sample is faint, the CCD camera 8 serving as the light receiver has a sensitivity as high as possible. In order to suppress the dark current generated from the CCD camera 8, a cooling device comprising a Peltier element is provided at the bottom of the CCD camera 8, and the temperature of the CCD camera 8 is cooled and kept at about 0 ° C. An infrared cut filter is disposed above the light receiving surface of the CCD camera 8 to block infrared rays that are background light. A TV camera is connected to the CCD camera through a signal cable, and a sample image is drawn on the TV monitor. The CCD camera may be a three-plate color camera so that a color illumination image can be obtained.

  Of course, the photodetector is not limited to a CCD camera, and for example, a CMOS image sensor or a SIT camera may be used.

Next, the structure of the weak light detection apparatus having means for stimulating cells according to the present invention will be described with reference to FIG. A test sample is put into a sample container 21 such as a petri dish together with a culture solution. A water tank 22 is disposed around the sample container 21, and pure water is supplied into the water tank 22 through a nozzle 23. This pure water is sent to maintain the humidity in the sample container. Further, CO ₂ gas is supplied to the upper surface of the water tank 22 through a gas supply tube 24. CO ₂ gas is supplied from a gas cylinder placed outside the main body of the measuring apparatus. See FIG. The gas cylinder is filled with a mixed gas of, for example, 5% CO _{2 and} 95% O ₂ . CO ₂ gas is supplied from a gas cylinder into a container containing a sample container through a gas supply tube at a flow rate of 50 mL / min. Further, the entire sample container is on the heat plate 27. The environmental temperature of the heat plate 27 is set at 0.5 ° step intervals by a temperature controller (not shown). The bottom surface of the sample container 21 is made of the same material as the microscope cover glass and has a thickness of 0.17 mm so that a normal objective lens can be used. The bottom surface of the sample container 21 is optically transparent. The sample container is not limited to a petri dish, and a slide glass, a microplate, or the like may be used.

  Furthermore, it is equipped with an automatic dispensing device 100 that is one of the means for stimulating cells, the solution is sucked from the reagent container 101 by the pump 102, the lid and the sample container lid are opened by a motor (not shown), and a predetermined amount is placed in the sample container. The reagent is discharged from the nozzle 23. When the discharge of the solution is completed, the lid and the sample container lid are closed by the motor. An illumination image, a luminescence image, a fluorescence image, and the like can be acquired in synchronization with a means for stimulating a cell and a means for acquiring an image.

  Two stepping motors (not shown) are separately attached to the sample stage 3 in the 90 ° direction, and each stepping motor is connected to a sample stage controller. The sample stage controller is connected to the computer 16, and in response to a command from the computer 16, the two stepping motors are driven to move the sample stage in the X direction and the Y direction.

  An objective lens 15 is arranged upside down below the sample stage, and an objective lens heater 28 is attached to the periphery in contact with the objective lens 15. The objective lens heater 28 is connected to a temperature adjusting device, temperature is controlled at 0.5 ° step intervals, and the objective lens is held at a constant temperature from the outside. An objective lens Z-axis drive mechanism is provided around the objective lens heater, and automatically drives the objective lens 15 along the Z-axis (optical axis direction). The objective lens Z-axis drive mechanism moves the objective lens up and down by a rack and pinion mechanism (not shown). The rotation of the knob of the rack and pinion mechanism is performed by a computer-controlled stepping motor (not shown). The objective lens Z-axis drive mechanism may be a friction roller mechanism in addition to the rack and pinion mechanism.

  When measuring a luminescence phenomenon caused by a bioluminescent protein, the luminescence intensity is still very weak at the time of sample setting, and almost cannot be detected as a light signal by a light receiver. Therefore, the internal structure of the cell cannot usually be observed. That is, the objective lens cannot be focused while confirming the cell that is the observation target in the sample, as in normal microscope observation. Therefore, as illumination image image observation, light from a light source such as a halogen lamp is projected onto a test sample through an illumination optical system to obtain an illumination image image of the microscope by the test sample, and based on this, the focus position of the objective lens is determined. decide. That is, in the illumination image observation, two substantially central positions on the optical axis of the objective lens that can obtain a high contrast image are set as the focus positions of the objective lens. Thereby, when the luminescence intensity by the bioluminescent protein becomes large, a clear luminescent image focused on the CCD camera can be obtained.

  Next, functions of the present embodiment will be described based on the block diagram of FIG. The image of the test sample is input to the image sensor 7 of the CCD camera 8, where it is converted into an analog electrical signal, that is, an image signal. The converted image signal is input to the signal processing circuit 33, where it is subjected to image processing such as amplification processing, filtering processing, and contour enhancement, and then guided to the digital zoom circuit 34. The image signal is A / D converted by the digital zoom circuit 34 to generate a digital image signal, and digital zoom processing is performed on the digital image signal in accordance with a digital zoom control signal supplied from the CPU 35. The processed digital image signal is input to the TV monitor 37 through the display control circuit 38, and an enlarged image of the test sample is displayed on the screen of the TV monitor 37.

  On the other hand, the digital image signal output from the digital zoom circuit is also input to the recording / playback control circuit 38. The recording / reproducing control circuit 38 records the input digital image signal on a removable recording medium 39 accommodated in a memory slot (not shown) based on the recording control signal supplied from the CPU 35. A memory card is used as a recording medium. The recording / reproducing control circuit 38 reads a digital image signal recorded on the recording medium 39 in response to a reproducing control signal given from the CPU 35. The read digital image signal is input to the TV monitor 37 via the display control circuit 38, whereby an image of the test object is displayed on the screen of the TV monitor 37.

  Note that image recording is not limited to a memory card, and may be recorded on a recording medium such as a hard disk, a magnetic disk, or a magneto-optical disk.

  Further, as an important configuration, the CPU is connected to the dispensing device and the illumination device, and is controlled so as to acquire an imaging signal from the CCD camera 8 in synchronization with the execution of the dispensing operation by the dispensing device. Yes.

  Further, an operation panel as shown in FIG. 5 is connected to the CPU. In response to an instruction from the operation panel, a digital zoom control signal is supplied to the digital zoom circuit via the CPU. At this time, the digital zoom control signal issued from the CPU is a rectangular continuous pulse signal. The operation panel also incorporates a recording button and a playback button. When the recording button is turned on, the CPU enters a recording mode, generates a recording control signal, and supplies it to the recording / reproducing control circuit. On the other hand, when the playback button is turned on, the CPU enters the playback mode, generates the playback control signal described above, and supplies it to the recording / playback control circuit. A control program for controlling the operation of the CPU is stored in the memory. By pressing these buttons, an instruction signal corresponding to each button is output from the operation board to the CPU to adjust the zoom center position, zoom magnification, and the like. In FIG. 5, the shutter button, the zoom enlarge button, the zoom reduce button, and the cross cursor button are shown as examples, and the description of the other operation members is omitted.

  As can be seen from the above description, the feeble light measuring apparatus of the first embodiment has a digital zoom function capable of adjusting the zoom magnification by digitally processing the acquired image signal. The zoom magnification M by the digital zoom function is 4.0 times at the maximum, and the zoom magnification M can be varied within a range of M = 1.00 to 4.00 by controlling the digital zoom circuit according to the digital zoom control signal described above. it can. Since a known technique is used for the digital zoom circuit that assumes the digital zoom function, the description thereof is omitted here.

  The digital signal processing unit performs digital processing such as gamma correction on the image data input from the analog signal processing unit (A / D converter) as necessary, and also performs CCD processing based on a control signal from the CPU. Is used as image capturing means for capturing the image data of the digital zoom area from the image data captured by (the image data of the entire pixel area of the CPU). That is, when a control signal for controlling the zoom magnification and the zoom center position is input from the CPU to the digital signal processing unit, the image data of the zoom magnification centered on the zoom center position is cut out and an image for one screen is obtained. This image can be generated and captured as an image in the digital zoom area.

In particular, in the digital signal processing unit, since the zoom center position and zoom magnification are visually shown to the operator, the captured image of the digital zoom area can be output to the display memory and displayed on the LCD. Thus, the photographer can grasp the zoom center position, and by pressing the cross cursor button, the zoom center position can be moved to an arbitrary position, and the zoom center position can be designated at a desired position. Also, by pressing the zoom enlarge / reduce button, the digital zoom area can be changed and a desired zoom magnification can be designated.

  The memory card records and stores image data. When the mode switch is set to the imaging mode and an image recording instruction is input by pressing the shutter button, a control signal is output from the CPU to the digital signal processing unit, and one frame of image data is read from the CCD. After the digital signal processing is performed in the processing unit, the image data is written in the memory. The image data written in the memory is compressed by the compression circuit, and recorded and stored in the memory card by the card reader / writer device.

 Zooming is not limited to digital zoom, and may be performed using optical zoom. Optical zoom is performed by a zoom lens. As usual, optical zoom is performed by moving the focal distance between the CCD camera and the second relay lens on the optical axis by a motor driven by a stepping motor. The zoom lens configuration consists of a three-magnification lens group, a correction lens group that corrects aberrations, and a focus lens that performs focus adjustment. The focal length of the lens can be changed manually or automatically in 10 steps. . The zoom lens is driven by operating a zoom motor attached around the zoom lens. As the zoom motor, an ultrasonic motor or the like is used, and the lens is moved along the optical axis based on a control signal issued from the CPU.

Example 2
Simultaneous measurement of fluorescence and bioluminescence FIG. 6 shows an outline of an optical system of a weak light detection apparatus capable of simultaneously performing digital zoom and optical zoom, and capable of simultaneously measuring fluorescence and bioluminescence.

  The configuration and operation of the optical system are basically described in the first embodiment of FIG. 2 except that an optical system for excitation light (excitation light source, collimating lens, deflection mirror, dichroic mirror) is added. Same as the contents. The basic configuration is based on an inverted optical microscope. A light source for illumination, a sample stage, a sample stage, an objective lens, and a computer are included. The optical system includes an illumination optical system that guides light to a test object in a sample container such as a petri dish, and an observation optical system that guides fluorescence emitted from the test object to a photodetector.

  The excitation optical system includes an excitation light source, a collimating lens, a deflection mirror, and a switchable dichroic mirror. As the excitation light source, a visible gas laser such as an argon laser or a helium neon laser is usually used. The switchable dichroic mirror has a spectral characteristic that reflects light of the oscillation wavelength of the excitation light source and transmits the fluorescence and the spectrum of the emission signal. An excitation light source is provided outside the main body. The excitation light source is an argon laser with a wavelength of 488 nm and an output of 10 mW. The laser light is converted into a circular parallel light beam having a beam width by a collimating lens, reflected by a deflecting mirror, and incident on a switchable dichroic mirror installed in an observation optical system. The laser light incident on the observation optical system is reflected by the switchable dichroic mirror, enters the objective lens from below, and is condensed and irradiated onto the test sample in the sample container. The switchable dichroic mirror is housed in a holder (not shown), and is installed in a replaceable manner according to the oscillation wavelength of the excitation laser light. If there is no need to change the wavelength of the excitation light source, a normal dichroic mirror may be fixed and used without using the switching dichroic mirror.

In order to obtain an illumination image, an objective lens having a numerical aperture of about NA (numerical aperture) 0.9 is used (in the air). For immersion samples, use a high NA objective lens of 1.0 or higher. The fluorescence signal and the emission signal emitted from the test sample pass through the objective lens again, pass through the observation optical system of the apparatus main body, and reach the switchable dichroic mirror. Then, the light passes through the switching dichroic mirror, passes through the first relay lens and the second relay lens, is reflected by the switching mirror, and is focused on the light receiving surface of the image sensor of the CCD camera. Light is received by the imaging surface of the CCD camera through the second and third relay lenses.
Similarly, the light emitted from the bioluminescent material passes through the optical system of the same apparatus body, is reflected by the switching mirror, and focuses on the imaging surface of the CCD camera.

    The fluorescence signal and the emission signal emitted from the test sample reach the eyepiece when the switching mirror is removed from the optical path, and the observer can directly observe the image.

  The optical signal from the CCD camera is sent to an external computer, and the computer displays and analyzes the luminescence image, performs time measurement of the luminescence intensity, and performs signal analysis. The analysis result is displayed on the computer monitor screen. That is, the computer not only renders a sample image, but also analyzes temporal changes in emission intensity.

    For example, rhodamine green (RhG) is used as the fluorescent dye. In addition to rhodamine green (RhG), for example, TMR (Tetramethylrhodamine), 5-Tamra (5-carboxytetramethylrhodamine), etc. may be used as the fluorescent substance. In this case, an argon laser having a wavelength of 514.5 nm may be used as an excitation laser light source to excite the fluorescent substance TMR, and a He / Ne laser having a wavelength of 543.5 nm may be used to excite the fluorescent substance 5-Tamra. Other fluorescent dyes such as FITC (Fluorescein-isothiocyanate), TOTO 1, Acridine-Orange, Texas-Red, etc. may be used.

  FIG. 7 shows a weak light detection apparatus that can perform digital zoom and optical zoom simultaneously, and can simultaneously measure fluorescence and bioluminescence. The main body of the measuring device is placed in a light-shielding light-shielding box and fixed on the bottom plate with a stopper. The light shielding box has a light shielding lid separated from the upper surface, and one end of the light shielding lid is connected to the light shielding box main body by a hinge so that the light shielding lid can be opened and closed in a fan shape.

For example, a halogen lamp or a metal halide lamp is used as an illumination light source, and illumination light is guided from the upper surface of the sample container on the sample stage through the illumination optical fiber to illuminate the entire test sample. In the observation optical system, the deflection mirror used in the first embodiment is not used, and the signal light condensed by the objective lens is directly received by the CCD camera.

  A laser light source is fixed inside the light source box outside the main body, and laser light emitted from the laser light source enters the apparatus main body through a laser incident port attached to the outer frame. As the laser, an argon laser having a wavelength of 488 nm and an output of 10 mW is used. The laser light incident on the apparatus main body through the laser incident port enters the observation optical system, is reflected by the switchable dichroic mirror, enters the objective lens from below, and is focused and irradiated on the test sample. The switchable dichroic mirror is housed in the holder. The switchable dichroic mirror mounted in the holder is installed in a replaceable manner according to the oscillation wavelength of the excitation laser light.

  A lens is arranged at the bottom of the lens barrel attached to the main body, and a CCD camera is placed so that the center of the light receiving surface is aligned with the focus position on the Z axis. Since the light emitted from the sample is weak, use a CCD camera with the highest sensitivity possible. The number of pixels of the CCD camera is 1,360 x 1,024. In order to suppress the dark current generated from the CCD camera, a cooling device composed of Peltier elements is attached to the bottom of the CCD camera, and the temperature of the CCD camera is cooled and kept at about 0 ° C. An infrared cut filter is installed above the light-receiving surface of the CCD camera to block infrared light as background light. However, when infrared light is extracted as signal light, the infrared cut filter is removed from the lower part of the lens barrel before measurement. A signal processing unit and then a TV monitor are connected to the output unit of the CCD camera through a signal cable, and a sample image is drawn on the TV monitor. The CCD camera may be a three-plate color camera so that a color illumination image can be obtained. The main frame can slide up and down the lens barrel.

The sample container is fixed on the XY sample stage with a stop pin. The position of the XY sample stage can be freely moved along the XY plane by a rack and pinion mechanism.
An objective lens is installed below the sample container. The objective lens is attached to an objective lens Z-axis drive mechanism and can move along the optical axis (Z-axis). The objective lens Z-axis drive mechanism is connected to a focus detection unit installed outside the weak light detection device by a conductive wire, connected to the position detection unit from the focus detection unit, and an output signal of the position detection unit is output to the TV monitor .

  The lens barrel is separated into an upper part of the lens barrel and a lower part of the lens barrel, and the upper part of the lens barrel and the lower part of the lens barrel are connected. Fluorescence or light emission signals emitted from the cells pass through the switchable dichroic mirror, pass through a lens attached to the bottom of the lens barrel, and are collected on the light receiving surface of the CCD camera. The apparatus main body is fixed to the main body frame. The main body frame is attached to the support column, and the main body frame can be moved up and down. The lower part of the lens barrel is fixed on the base frame by a stopper. The support and base frame are fixed on the bottom plate.

  Next, the operation will be described. A signal of the illumination image of the test sample obtained from the CCD camera by the illumination light source is guided to the signal processing unit, and an optical signal intensity distribution is obtained as statistical processing data composed of output intensity signals from each pixel. In conjunction with the objective lens Z-axis drive mechanism, the objective lens Z-axis drive mechanism is operated to move the objective lens by an appropriate amount along the optical axis. The signal processing unit analyzes the light output signal from the CCD camera at each objective lens movement step, and detects the position of the objective lens on the optical axis where a high-contrast image is obtained. The position on the optical axis of the objective lens from which two high and low high-contrast images are obtained is found, and calculation is performed by the signal processing unit, so that the approximate center position of the two upper and lower positions is the light emission position of the test sample. Subsequently, the objective lens Z-axis drive mechanism is operated to move the objective lens to this position.

Now that the focus position of the objective lens has been determined, the objective lens is fixed at this position, and a fluorescence signal and a light emission signal emitted from the test sample are received by the CCD camera.
The objective lens is interchangeably attached to the objective lens Z-axis drive mechanism. When the magnification of the objective lens is increased and the test sample is imaged, the obtained image becomes dark. Test samples such as cells move in the culture medium. When this is observed with a high-magnification objective lens, the field of view becomes narrow, and in some cases, the test sample may move and disappear from the field of view. Accordingly, first, the test sample is observed with a low-magnification objective lens such as x10 or x20. Then, the desired position of the test sample is confirmed and designated using a mouse or keyboard, and the zoom is increased. A computer may be used as the signal processing unit.

Example 3
In this example, using a weak luminescence measuring device with an automatic dispensing device added to the device of Example 1, targeting a plurality of HeLa cells introduced with a luciferase gene, specific HeLa cells caused by drug stimulation We examined the measurement method to observe the emission intensity over time and observe the emission intensity.

The luciferase gene is transferred to the vector "pcDNA6 / TR (manufactured by Invitrogen)" that constantly expresses the tetracycline repressor (TetR) and the expression vector "pcDNA4 / TO (manufactured by Invitrogen)" with the tetracycline operator (TetO2). A specimen in which HeLa cells are co-expressed with the connected plasmid is used as an imaging target of this example. In this state, as shown in FIG. 8, since the TetR homodimer is bound to the TetO2 region, transcription of the luciferase gene is suppressed. Next, as shown in FIG. 9, tetracycline is added to the culture medium to bind to the TetR homodimer, and by changing the three-dimensional structure of the TetR homodimer, the TetR homodimer is separated from TetO2 to induce the transcription of the luciferase gene. To do. The culture medium is a D-MEM medium containing 10 mM HEPES and contains 1 mM luciferin.

The lens used as the objective lens and imaging lens is a commercially available microscope objective lens with specifications of Olympus UApo / 340, 20x / 0.75 “Oil, 20 times, NA 0.8” and “5 times, NA 0.13”. Yes, the total magnification corresponding to the magnification Mg is 4 times. The camera used is a digital camera “DP30BW (manufactured by Olympus)” cooled at 5 ° C., and the CCD element is 2/3 inch type, the number of pixels is 1360 × 1024, and the pixel size is 6 μm square. In the present embodiment, imaging of HeLa cells is performed in a state where the entire apparatus to be used is covered with a dark box.

Next, an experimental protocol in this example will be described.
A sample is prepared by co-expressing a vector in which TetR is constitutively expressed and a plasmid in which a luciferase gene is linked to an expression vector having TetO2 in HeLa cells.
The specimen of (1) above is taken as an imaging target, and an illumination image and a luminescent image are taken. The illumination image and the light emission image are always taken as a pair. The illumination image is captured with an exposure time of 10 msec, and the luminescent image is captured with an exposure time of 5 min.
Tetracycline is added to the specimen to induce transcription of the luciferase gene in the specimen.
Imaging is performed immediately after the addition of tetracycline. Note that, similarly to the above (2), an illumination image and a light emission image are taken as a pair.
After the first imaging, the above imaging (2) is repeated. It is assumed that there is a 10 min interval between each imaging. FIG. 10 shows a graph of the timing of addition of tetracycline and exposure of the illumination image / luminescence image in the above (2), (3), (4), (5).
After the addition of tetracycline, the above operation (5) is repeated until 10 hours later.
Imaging in the above (2), (4), (5), and (6) is automatically performed by computer processing. The optical signals obtained in the above (2), (4), (5), and (6) are automatically converted into digital data.
The acquired illumination image and luminescence image are superimposed to identify HeLa cells in which luciferase is expressed. An illumination image is shown in FIG. 11, a light emission image is shown in FIG. 12, and a superimposed image is shown in FIG. When it is difficult to discriminate the light emitting site of the HeLa cell in the superimposed image, the output value from the CCD camera may be displayed in a pseudo color to make it easier to visually recognize.
Changes in luminescence intensity over time in HeLa cells expressing luciferase are digitized and analyzed.

  FIG. 14 shows a graph of the temporal variation of the luminescence intensity of the HeLa cells obtained by the protocol of this example described above. According to the present example, it was proved that it is possible to specify which cell emits light and to measure the temporal change of light emission.

Example 4
In this example, using the same apparatus as in Example 3, for a luminescent bacterium (P. phosphoreum) that is a sample that moves with the passage of time, a measurement technique that tracks changes in luminescence intensity over time due to drug stimulation Was examined.

  Luminous bacteria are long-known luminescent organisms: (i) independent living in seawater, (ii) symbiosis in the luminaries of fish and cephalopods, (iii) breeding on dead fish, animal meat, (iv) It exists widely in the natural world in the form of habitual parasitism in the digestive tract of saltwater fish and the skin of squid. The luminescence pattern of the luminescent bacteria under aerobic conditions is continuous luminescence, which is strong enough to be observable with the naked eye.

  The objective lens in this example was Olympus UApo / 340, 40x / 1.35 Oil Iris “Oil, 40 ×, NA 1.35”. Other imaging conditions were taken under the same conditions as in Example 3.

The experimental protocol of this example is shown below.
Isolate luminescent bacteria symbiotic to the firefly squid luminophore and culture in LB medium with 3% NaCl concentration. Glycerol is added to this culture solution to prepare a specimen having a final concentration of 25% glycerol solution (v / v).
The specimen of (1) above is taken as an imaging target, and an illumination image observation image and a light emission image are taken. The illumination image and the light emission image are always taken as a pair. The illumination image observation image is captured with an exposure time of 10 msec, and the light emission image is captured with a exposure time of 1 min.
Using an automatic dispenser, Ampicillin is added to the specimen to inhibit the cell wall synthesis of luminous bacteria. As a result, the luminescent bacteria are inhibited from growing and are killed.
The automatic dispenser and the means for acquiring images are synchronized, and imaging is performed immediately after the addition of Ampicillin. Note that, as in the above (2), an illumination image and a light emission image are always taken as a pair.
After the first imaging, the above imaging (2) is repeated. It is assumed that there is a 5 min interval between each imaging.
After adding Ampicillin, repeat step (5) until 1 hour later.
Imaging in the above (2), (4), (5), and (6) is automatically performed by computer processing. The optical signals obtained in the above (2), (4), (5), and (6) are automatically converted into digital data.
The acquired illumination image and luminescence image are superimposed to identify the luminescent bacteria that emit intense light. An illumination image observation image is shown in FIG. 15, and a light emission image is shown in FIG. FIG. 17 shows a superimposed image of the illumination image and the light emission image colored with pseudo color.
The superimposed images are arranged in order of time and animated to track changes in the luminescent position as the luminescent bacteria move. The luminescent bacteria may be traced automatically by computer processing.
Quantify and analyze the time-dependent variation of luminescence of luminescent bacteria.

    FIG. 18 shows a graph of the variation with time of the luminescence intensity of the luminescent bacteria obtained by the protocol of this example described above. According to this example, it was proved that even if microorganisms such as bacteria move during the measurement, it is possible to specify which microorganisms emit light and to measure the change in light emission over time.

As mentioned above, although this invention was demonstrated by embodiment, all the inventions derived | led-out from the description mentioned above are included, and it extends to the equivalent. Further, the present invention is not limited to the above-described embodiments, and various modifications are possible, and these designs, products, or methods of use are included.

It is a figure which shows a measurement procedure. It is the schematic of the weak light detection apparatus of this invention based on an inverted microscope. It is a figure which shows the structure of the weak light detection apparatus which has a means to give a stimulus to a cell. 3 is a block diagram illustrating functions of Embodiment 1. FIG. 2 is a diagram illustrating an operation panel in the apparatus described in Embodiment 1. FIG. FIG. 3 is a diagram schematically illustrating an optical system of a weak light detection apparatus that performs simultaneous measurement of fluorescence and bioluminescence described in Example 2. It is a figure which shows the weak light detection apparatus which performs the measurement about fluorescence and bioluminescence, and the image expansion by digital zoom and optical zoom simultaneously, respectively. FIG. 4 is a schematic diagram of a plasmid in which a luciferase gene is linked to an expression vector having a tetracycline operator (TetO2) in Example 3. FIG. 3 is a schematic diagram showing a state where TetR homodimer is separated from TetO2 by the action of tetracycline. It is a figure which shows the addition timing of the tetracycline in Example 3, and the exposure timing of an illumination image and a light emission image. It is a figure which shows the illumination image acquired in Example 3. FIG. It is a figure which shows the illumination image acquired in Example 3. FIG. It is a figure which shows the image which overlap | superposed the illumination image acquired in Example 3, and the light emission image. 4 is a graph showing temporal changes in luminescence intensity of HeLa cells in Example 3. It is a figure which shows the illumination image acquired in Example 4. FIG. It is a figure which shows the illumination image acquired in Example 4. FIG. It is a figure which shows the image which overlap | superposed the illumination image acquired in Example 4, and the light emission image. It is a graph of the time-dependent fluctuation | variation of the luminescence intensity of the luminescent bacteria in Example 4.

Explanation of symbols

1? ? ? Illumination optics 3? ? ? Sample stage 2? ? ? Light source 4? ? ? Test sample 5? ? ? Observation optical system 8? ? ? CCD camera 9? ? ? Objective lens Z-axis drive mechanism 15? ? ? Objective lens 19? ? ? Imaging surface 21? ? ? Sample container 22? ? ? Aquarium 27? ? ? Heat plate 30? ? ? Sealed container 34 with lid? ? ? Digital zoom circuit 35? ? ? CPU
37? ? ? TV monitor 38? ? ? Recording / reproduction control circuit 40? ? ? Operation panel 51? ? ? Collimating lens 53? ? ? Switchable dichroic mirror 54? ? ? Shading box 61? ? ? Excitation light source

Claims (13)

A method of acquiring an image of a sample using an optical imaging means,
Illuminating a test sample and obtaining an illumination image;
Determining a focus position of an objective lens corresponding to a light emission position of the test sample using the illumination image;
A step of acquiring a luminescence image of a cell without illuminating the test sample at the luminescence position,
Superimposing the illumination image and the luminescence image of the cell;
The process of specifying the analysis area,
A method for obtaining an image of a test sample derived from an organism using an optical imaging means. A method for obtaining an image of a test sample derived from a living organism according to claim 1 using an optical imaging means.
After the step of superimposing the illumination image and the light emission image of the cell,
A method for obtaining an image of a test sample derived from a living organism using an optical imaging means, wherein the superimposed image is displayed or recorded. A method for obtaining an image of a test sample derived from an organism according to claim 1 or 2 using an optical imaging means,
Furthermore, it has the process of acquiring the fluorescence image of a cell, The method of acquiring the image of the test sample derived from the organism using an optical imaging means characterized by the above-mentioned. In the method of acquiring the image of the test sample derived from the organism according to any one of claims 1 to 3 using an optical imaging means,
A method for acquiring an image of a test sample derived from a living organism using an optical imaging means, comprising illuminating a test sample and acquiring an illumination image a plurality of times. In the method of acquiring the image of the test sample derived from the organism according to any one of claims 1 to 4 using an optical imaging means,
Before or after the process of stimulating cells
Illuminating a test sample and obtaining an illumination image;
A step of acquiring a luminescence image of a cell without illuminating a test sample;
A method for obtaining an image of a test sample derived from an organism using an optical imaging means. A method for obtaining an image of a test sample derived from a living organism according to claim 5 using an optical imaging means,
A method for obtaining an image of a test sample derived from an organism using an optical imaging means, wherein the step of stimulating cells is by administration of a drug, electrical stimulation, gas, or heat. In a method for obtaining an image of a test sample derived from an organism according to claim 1 using an optical imaging means,
Furthermore, a step of measuring the light intensity in the analysis region,
A method for acquiring an image of a test sample derived from an organism having a step of displaying the change in light intensity using an optical imaging means. The method of obtaining an image of a biological test sample according to claim 7 using an optical imaging means,
A method of acquiring an image of a test sample derived from a living organism using an optical imaging means, wherein at least two of an illumination image of a designated region, a luminescence image of a cell, and a fluorescence image of a cell are displayed. An apparatus for acquiring an image of a biological test sample using an optical imaging means,
Means for illuminating the test sample;
Means for obtaining an illumination image;
Means for determining a focus position of an objective lens corresponding to a light emission position of the test sample using the illumination image;
Means for obtaining a luminescence image of a cell at the luminescence position;
Means for superimposing the illumination image and the luminescence image of the cell, and means for designating an analysis region;
An apparatus for acquiring an image of a biological test sample using an optical imaging means. An apparatus for acquiring an image of a test sample according to claim 9 using an optical imaging means,
Furthermore, an apparatus for acquiring an image of a test sample derived from a living organism using an optical imaging means, characterized by having means for displaying or recording the superimposed image. In an apparatus for obtaining an image of a test sample according to claim 9 or 10 using an optical imaging means,
Furthermore, an image of a test sample derived from an organism characterized by having means for stimulating cells,
An apparatus that acquires using optical imaging means. An apparatus for acquiring an image of a test sample according to claim 11 using an optical imaging means.
An optical imaging means is used for an image of a test sample derived from a living body, wherein the means for stimulating cells is at least one of a reagent supply means, a temperature adjustment means, and a gas supply means. Device to acquire. In an apparatus for obtaining an image of a test sample according to any one of claims 9 to 12 using an optical imaging means,
Furthermore, the apparatus which acquires the image of the test sample derived from the living body characterized by having a means to acquire the fluorescence image of a cell using an optical imaging means.