JP5124216B2 - Optical signal observation method and optical signal observation system - Google Patents

Description

  The present invention relates to an optical signal observation method and an optical signal observation system for observing an optical signal emitted from a photosensitive organism.

  Conventionally, as a technique for examining gene expression in photosensitive organisms such as plants and cyanobacteria, a technique related to a reporter assay using a photoprotein such as luciferase as an index is known (see, for example, Patent Documents 1 and 2). In this reporter assay, the total amount of luminescence from the whole plant is generally detected by using a luminometer.

JP 2002-310894 A JP-T-2004-506879

  However, in the case of plants, it is expected that the gene expression pattern will be different for each part such as roots, stems, leaves, etc. in the process of long-term development and growth, and the amount of luminescence accompanying it is also different for each part and further for each cell. Expected to be different. For this reason, in the above-described conventional technology, it has been difficult to stably observe light emission at the cellular level of a photosensitive organism over a long period of time.

  The present invention has been made in view of the above, and an object of the present invention is to provide a light signal observation method and a light signal observation system capable of stably performing light emission observation at the cellular level of a photosensitive organism over a long period of time. And

  In order to solve the above-described problems and achieve the object, the optical signal observation method according to the present invention is a photosensitivity organism that has photosensitivity and is labeled so as to emit an optical signal according to biological activity or behavior. An optical signal observation method for observing an emitted optical signal, the stimulating light irradiation step for irradiating the photosensitive organism with stimulation light having a component that the photosensitive organism senses based on a predetermined condition, and the stimulation light irradiation A luminescence image imaging step for imaging a luminescence image formed by imaging a light signal emitted by the photosensitive organism irradiated with the stimulation light in a step, and the luminescence image imaging step after the stimulation light irradiation step is completed. An interval is provided before starting.

  The optical signal observation method according to the present invention is characterized in that, in the above invention, the interval time is determined based on a temporal change of phosphorescence generated around the photosensitive organism.

  The light signal observation method according to the present invention is the light emission image according to the above invention, wherein the light emission image captured in the light emission image capturing step is subjected to correction based on a temporal change of phosphorescence generated around the photosensitive organism. It further has a correction step.

  The optical signal observation method according to the present invention is characterized in that, in the above-described invention, the stimulation light irradiation step and the luminescent image capturing step are repeatedly performed under a predetermined condition.

  Further, the optical signal observation method according to the present invention is the above-described invention, wherein the bright-field image of the photosensitive organism is irradiated with light for bright-field observation after the stimulation light irradiation step and before the interval. An image obtained by superimposing a bright-field image captured in the bright-field image capture step, a bright-field image captured in the bright-field image capture step, and a light-emitting image first captured in the light-emitting image capture step after capturing the bright-field image. An image generation step to be generated, and an output step to output a superimposed image of the bright field image generated in the image generation step and the light emission image are further provided.

  The optical signal observation method according to the present invention is characterized in that, in the above invention, labeling is performed by introducing a gene comprising a photoprotein into the photosensitive organism.

  The optical signal observation method according to the present invention is characterized in that, in the above invention, the photoprotein is luciferase.

  An optical signal observation system according to the present invention is an optical signal observation system for observing an optical signal emitted by a photosensitive organism having photosensitivity and labeled so as to emit an optical signal according to biological activity or behavior, Stimulation light irradiation means for irradiating the light-sensitive organism with stimulation light having a component sensed by the light-sensitive organism, and light emission for imaging a light emission image formed by imaging a light signal emitted by the light-sensitive organism Image capturing means, and control means for performing control to provide an interval between the time when the stimulation light irradiation means ends the irradiation of the stimulation light and the time when the light emission image imaging means starts capturing the light emission image. It is characterized by that.

  According to the present invention, after irradiating the photosensitive organism with stimulation light having a component that the photosensitive organism senses based on a predetermined condition, an interval is provided, and then a light signal from the photosensitive organism is imaged. By capturing a luminescent image, background noise can be reduced and a weak light signal can be detected with high sensitivity. Therefore, it is possible to stably observe the luminescence at the cellular level of the photosensitive organism over a long period of time.

  The best mode for carrying out the present invention (hereinafter referred to as “embodiment”) will be described below with reference to the accompanying drawings.

(Embodiment 1)
FIG. 1 is a diagram schematically showing an overall configuration of an optical signal observation system according to Embodiment 1 of the present invention. The optical signal observation system 1 shown in the figure can transmit and receive data between the observation device 2 and the observation device 2 that optically observes the optical signal emitted from the sample Sp, which is a photosensitive organism, as image data. And a data processing device 3 that performs drive control of the observation device 2 and performs arithmetic processing using image data sent from the observation device 2, and these two devices cooperate to emit light of the sample Sp. Make observations.

  Here, the sample Sp used in the first embodiment will be described. The sample Sp is a photosensitive organism having photosensitivity such as a plant or cyanobacteria. A gene consisting of a photoprotein is introduced into the photosensitive organism constituting the sample Sp. In the first embodiment, luciferase is introduced as a gene consisting of a photoprotein. When introducing luciferase into a cell, a method of directly introducing it by a particle gun method, an electroporation method or the like, or a method of using a transformed plant can be employed.

  In addition, luciferin, which is a luminescent substrate, is introduced into the sample Sp. As a method for introducing luciferin, there are a method in which a luciferin solution is directly sprayed on a site to be observed and a method in which luciferin is added to an agar medium. Among these, in the method of adding luciferin to an agar medium, it is known that luciferin absorbed from the root of a plant penetrates to the leaves and stems of the plant.

  When luciferase is introduced into a cell as a reporter gene and the intensity of gene expression, which is an example of biological activity, is examined using luciferase activity as an indicator, the DNA fragment is obtained by connecting the target DNA fragment upstream or downstream of the luciferase gene. The effect on transcription can be investigated. In addition, by connecting a gene such as a transcription factor that is thought to affect transcription to an expression vector and co-expressing it with the reporter gene, the effect of the gene product on the expression of the reporter gene can be examined.

  Hereinafter, the configuration of the optical signal observation system 1 will be described. First, the observation apparatus 2 includes a container 21 for storing the sample Sp, a movable stage 22 on which the container 21 is disposed, and a light-emitting image capturing unit that captures a light-emitting image formed by forming an image of a light signal emitted from the sample Sp. 23, a stage driving unit 24 that drives the stage 22 two-dimensionally, a transmission illumination unit 25 that irradiates the sample Sp with light for bright field observation (for example, white light), a light-emitting image capturing unit 23, and a transmission illumination unit And an optical path switching drive unit 26 for switching each of the 25 optical paths.

  As the container 21, in addition to a petri dish, a slide glass, and a microplate, a gel support, a fine particle carrier, and the like can be applied. In the case shown in FIG. 1, a photosensitive organism as a sample Sp grown on an agar medium K containing luciferin is cultured in a container 21 made of petri dish.

  The luminescence image pickup unit 23 is realized by using an upright luminescence microscope, and includes an objective lens 231, an imaging lens 232 that forms an image incident through the objective lens 231, and a luminescence image of the sample Sp. An imaging device 233 that captures a bright field image, a light source 234 that irradiates the sample Sp with stimulation light, a shutter 235 that switches irradiation and non-irradiation of the stimulation light from the light source 234, an objective lens 231, and an imaging lens 232 and a mirror 236 that is detachably disposed between the shutter 235 and the shutter 235.

As an optical condition for the imaging optical system including the objective lens 231 and the imaging lens 232 to image extremely weak light emission, when the projection magnification is β, (NA / β) ² is 0.01 or more. It is desirable to combine the objective lens 231 and the imaging lens 232 in such a manner.

  The imaging device 233 includes a solid-state imaging device such as a CCD or CMOS, generates image data by photoelectrically converting an image formed on the imaging surface of the solid-state imaging device, and outputs the image data to the data processing device 3. . As the solid-state imaging device included in the imaging device 233, a high-sensitivity monochrome CCD, for example, a cooled CCD of 0 ° C. or lower can be used, preferably a cooled CCD of −80 to −30 ° C., more preferably. A cooled CCD of about −60 ° C. may be used.

  The light source 234 irradiates the sample Sp with stimulation light for plant growth that irradiates the sample Sp, and a high-pressure sodium lamp or an LED lamp is used. In plant growth, blue light and red light are particularly important. For this reason, the sample Sp may be irradiated with light having a desired wavelength component by providing a spectral filter that splits the light emitted from the light source 234.

  The mirror 236 is moved by the optical path switching drive unit 26 under the control of the data processing device 3. Specifically, when the sample Sp is irradiated with the stimulation light from the light source 234, the mirror 236 is inserted on the optical path of the luminescent image capturing unit 23. On the other hand, when the imaging device 233 captures a luminescent image, the mirror 236 is detached from the optical path of the luminescent image capturing unit 23. The mirror 236 may be moved manually.

  The transmitted illumination unit 25 includes a light source 251 such as a halogen lamp that emits light for bright field observation, a shutter 252 for switching between irradiation and non-irradiation of light for bright field observation, and white light from the light source 251 on the sample Sp. An illumination lens system 253 for condensing light is provided, and is disposed on the opposite side of the light emission image capturing unit 23 with respect to the sample Sp.

  The illumination lens system 253 includes a collector lens 253a and a condenser lens 253b, and performs critical illumination on the sample Sp. Note that the illumination lens system 253 may perform Kohler illumination on the sample Sp.

  Next, the functional configuration of the data processing device 3 will be described with reference to the block diagram shown in FIG. The data processing device 3 is realized by a transmission / reception unit 31 that receives information including image data from the observation device 2 and transmits information including a control signal to the observation device 2, a keyboard, a mouse, and the like. The control unit 33 controls the operation of the optical signal observation system 1 while performing calculations based on the information received from the observation device 2 and the input unit 32 from which information such as measurement conditions and various operation instruction signals are input from the outside. And a storage unit 34 that stores image data and information including calculation results in the control unit 33, and an output unit 35 that outputs information including calculation results in the control unit 33.

  The control unit 33 determines whether or not a predetermined condition is satisfied in the process performed by the light emission amount measurement unit 331 that measures the light emission amount (light emission intensity) of the cell based on the image data and the optical signal observation system 1. A condition determination unit 332 that performs the determination, an image generation unit 333 that generates an image to be displayed by the output unit 35 based on the image data input from the observation device 2 and the light emission amount measured by the light emission amount measurement unit 331, and a timer And a watch 334 having a function. The control unit 33 is realized using a CPU having a calculation function and a control function.

  The storage unit 34 includes an image data storage unit 341 that stores the image data generated by the image generation unit 333, a measurement condition storage unit 342 that stores measurement conditions of the sample Sp input from the input unit 32, and a light emission amount measurement unit. And a light emission amount storage unit 343 that stores the light emission amount measured by 331. The storage unit 34 uses a ROM that stores in advance the processing according to the first embodiment and a program for starting a predetermined OS, a RAM that temporarily stores information used when the control unit 33 performs calculations, and the like. And at least a part of the storage means. The storage unit 34 may further include an auxiliary storage device that can read information recorded on a recording medium such as a hard disk, a flexible disk, a CD-ROM, a DVD-ROM, an MO disk, or a PC card.

  The output unit 35 has a function of generating an image based on a control signal from the control unit 33 and displaying the generated image, and includes a display such as liquid crystal, plasma, and organic EL. The output unit 35 may include a printer capable of outputting information using paper or the like as a medium.

  FIG. 3 is a flowchart showing an outline of processing of the optical signal observation method according to the first embodiment. FIG. 4 is a time chart showing the flow of temporal processing of the optical signal observation method according to the first embodiment. Hereinafter, the optical signal observation method according to the first embodiment will be described with reference to these drawings. It is assumed that the light source 234 of the luminescent image capturing unit 23 and the light source 251 of the transmissive illumination unit 25 are lit in advance, and the lighting state is maintained during a series of processing.

  First, the data processing device 3 receives an input of measurement conditions from the input unit 32 and stores it in the measurement condition storage unit 342 of the storage unit 34 (step S1). The measurement conditions are conditions at the time of photographing in the observation apparatus 2, conditions for culturing the sample Sp, and the like. When accepting an input in step S1, the output unit 35 may display a predetermined condition input screen and prompt the user to select and input various conditions.

  In the first embodiment, it is assumed that long-term observation of the sample Sp is performed. For this reason, it is necessary to irradiate growth light with respect to the sample Sp. In higher plants, day length (sunshine) time plays an extremely important role in regulating growth, development, cell differentiation, etc., and it is extremely important to appropriately control the time of light irradiation according to the observation content. is there. Therefore, in the first embodiment, an environment that can withstand long-term observation of the sample Sp is provided by inputting the light irradiation time for plant growth as measurement conditions in advance.

  Thereafter, the observation device 2 switches each light path of the light emission image capturing unit 23 and the transmission illumination unit 25 to a light path for stimulating light irradiation (step S2). Specifically, the optical path switching drive unit 26 opens the shutter 235 of the light emitting image capturing unit 23 and the mirror 236 with respect to the light emitting image capturing unit 23 in accordance with a control signal transmitted from the control unit 33 of the data processing device 3. While the shutter 252 of the transmission illumination unit 25 is closed.

As a result of step S2, the stimulation light emitted from the light source 234 is reflected by the mirror 236 and irradiated onto the sample Sp. In this way, the observation apparatus 2 irradiates the sample Sp with the stimulation light for the predetermined irradiation time T _L (step S3). The irradiation time T _L is set in step S1. Note that FIG. 4 illustrates a case where the irradiation time T _L when the stimulation light is repeatedly irradiated is always the same, but this is merely an example. For example, the irradiation time of the stimulation light may be changed for each cycle.

  Thereafter, the observation apparatus 2 switches the light paths of the light emission image capturing unit 23 and the transmission illumination unit 25 to light paths for bright field image capturing (step S4). Specifically, the optical path switching drive unit 26 closes the shutter 235 of the light emitting image capturing unit 23 and the mirror 236 with respect to the light emitting image capturing unit 23 in accordance with a control signal transmitted from the control unit 33 of the data processing device 3. The shutter 252 of the transmissive illumination unit 25 is opened while being separated from the optical path. As a result, the bright field observation light emitted from the light source 251 passes through the sample Sp and enters the imaging device 233 via the objective lens 231 and the imaging lens 232.

  The imaging device 233 captures a bright field image according to the control signal transmitted from the control unit 33. Image data captured by the imaging device 233 is transmitted to the data processing device 3. The data processing device 3 stores and stores the image data of the bright field image captured by the imaging device 233 in the image data storage unit 341 of the storage unit 34 together with information on the imaging time (step S5). The imaging region is set when the bright field image is captured by the stage driving unit 24 driving the stage 22 based on the control signal received from the data processing device 3. Note that the stage 22 may be manually moved by the user.

  FIG. 5 is a schematic diagram schematically illustrating a bright field image captured by the imaging device 233. In the figure, one leaf L and stem S of sample Sp are in the field of view, but the other leaf indicated by the dotted line is in an out of focus state.

  Subsequently, the observation device 2 switches each light path of the light emission image capturing unit 23 and the transmission illumination unit 25 to a light path for the light emission image (step S6). Specifically, the optical path switching drive unit 26 closes the shutter 252 of the transmitted illumination unit 25 in accordance with a control signal transmitted from the control unit 33 of the data processing device 3. As a result, light emitted from the light source 251 in addition to the light source 234 is no longer applied to the sample Sp, and the imaging device 233 is in a state where it can capture light emitted from the sample Sp.

  Thereafter, the optical signal observation system 1 stands by until a predetermined time T elapses after the shutter 235 is closed and the interval time measured by the clock 334 of the control unit 33 (No in step S7). The interval time T is provided to quench the background noise of the transmitted light. A glass device such as the container 21 contains a substance having afterglow called a phosphorescent substance. For this reason, when detecting a light signal with weak intensity using luciferase as an index, afterglow (phosphorescence) by the phosphorescent substance after irradiation with stimulating light may be effective as background noise. Therefore, in the first embodiment, an interval is provided until the background noise reaches a level that can be ignored. Depending on the type of photosensitive organism and the content of observation, it may not be necessary to capture a bright field image. In this case, an interval may be started immediately after the stimulation light is irradiated.

  The interval time T can be set as the time until the measured value falls below a certain threshold value. This threshold value is determined based on a dimming function obtained as a result of measuring a temporal change in the intensity of phosphorescence under the same conditions. This dimming function varies depending on the material of the base material and the like, but is a function that decreases exponentially with time.

  The interval time T set in this way is stored in the measurement condition storage unit 342, and a step is performed by comparing the elapsed time with the interval time T after the condition determination unit 332 finishes transmitting light. The determination process in S7 is performed.

  Thereafter, when the interval time T has elapsed (step S7, Yes), the imaging device 233 captures a light emission image of the sample Sp and transmits image data of the captured light emission image to the data processing device 3. The data processing device 3 writes and stores the received image data of the luminescent image in the image data storage unit 341 together with the time information of the imaging time (step S8). When capturing a luminescent image in step S8, it is necessary to take a sufficiently long exposure time compared to capturing a bright field image. FIG. 6 is a diagram schematically showing a light emission image. In the luminescent image P2 shown in the figure, luminescence of luciferase at the cellular level appears as a dotted region C.

  Next, the light emission amount measuring unit 331 measures the light emission amount (light emission intensity) for each location of the sample Sp using the image data of the light emission image stored in the image data storage unit 341, and positional information (in the imaging region) It is written and stored in the light emission amount storage unit 343 together with the time information and the like (identified by coordinates or the like) (step S9).

Thereafter, the condition determination unit 332 determines whether or not the state after the completion of step S9 satisfies a predetermined image generation condition (step S10). The image generation conditions here are defined using, for example, any one of the following conditions (1) to (3).
(1) The number of times of emission image capturing reaches a predetermined number.
(2) The light emission amount of the light emission image is larger than a predetermined value.
(3) A certain period of time has elapsed from the time when the first emission image was captured.

As a result of the determination by the condition determination unit 332, when the state after step S9 ends does not satisfy the image generation condition (No in step S10), the optical signal observation system 1 returns to step S2 and repeats the process. When this repetition is performed, it is possible to change the irradiation time T _L of the stimulation light according to the measurement conditions. Further, it is possible to change the wavelength component of the stimulation light or not to irradiate the stimulation light without changing the irradiation time _TL of the stimulation light.

  On the other hand, as a result of the determination by the condition determination unit 332, when the state after step S9 ends satisfies the image generation conditions (step S10, Yes), in the data processing device 3, the image generation units 333 of the control unit 33 are mutually connected. The corresponding bright field image and light emission image are read from the image data storage unit 341, and an image is generated by superimposing the read bright field image and light emission image (step S11).

  Thereafter, the output unit 35 displays the superimposed image generated by the image generation unit 333 (step S12). FIG. 7 is a schematic diagram showing a display output example (first example) of an image obtained by superimposing a bright-field image and a light-emitting image. The superimposed image P3 shown in the figure schematically shows a state in which light is emitted from the cells of the leaf L of the sample Sp, and the bright field image P1 shown in FIG. 5 and the luminescent image P2 shown in FIG. 6 are overlaid. It corresponds to the alignment. It should be noted that a pseudo color may be given to the punctiform region C corresponding to the cell, which is the light emitting portion of the reporter in the superimposed image P3.

  FIG. 8 is a schematic diagram illustrating a display output example (second example) in the output unit 35 of an image obtained by superimposing the bright field image and the light emission image. FIG. 8 schematically illustrates a case where the output unit 35 displays an animation of the superimposed images P3 and P4 of the bright field image and the light emission image in time series according to the acquisition time of the light emission image. As described above, the output unit 35 performs animation display, so that the user can visually grasp the temporal change in the light emission amount for each cell of the sample Sp.

  According to Embodiment 1 of this invention demonstrated above, after irradiating the said photosensitive organism | stimulation light which has a component which a photosensitive organism senses to the said photosensitive organism | system | group based on predetermined conditions, an interval is provided, and a photosensitive organism | organism | organism is carried out after that. By capturing a light-emitting image formed by imaging a light signal from the light source, background noise can be reduced and a weak light signal can be detected with high sensitivity. Therefore, it is possible to stably observe the luminescence at the cellular level of the photosensitive organism over a long period of time.

  Further, according to the first embodiment, it is possible to examine more detailed changes in gene expression by analyzing gene expression conventionally captured in the whole photosensitive organism at the site or cell level.

  In addition, according to the first embodiment, since a photoprotein is applied as a reporter gene, it is possible to perform observation with excellent quantification and suppressing damage to cells as much as possible. In particular, when luciferase is used as a reporter gene, luciferase is a chemically-excited luminescent enzyme that can be observed without irradiating excitation light, so it does not damage the sample and can last for several days to several weeks. Observation is possible.

  Moreover, according to this Embodiment 1, since it observes with an upright microscope, it is suitable for the three-dimensional observation of the photosensitive organism which grows in the vertical direction.

  In the first embodiment, a correction that reduces the influence of phosphorescence on the data processing apparatus side may be added to the luminescent image captured by the luminescent image capturing unit. In this case, by using the above-described dimming function, it is possible to correct the image by reducing the intensity of phosphorescence. Thereby, it is possible to shorten the interval time from the end of the irradiation of the light for bright field observation to the time of capturing the luminescent image, and to increase the exposure time at the time of capturing the luminescent image accordingly.

(Embodiment 2)
FIG. 9 is a diagram schematically showing the overall configuration of the optical signal observation system according to Embodiment 2 of the present invention. The optical signal observation system 4 shown in the figure can transmit and receive data between the observation device 5 and the observation device 5 that optically observes the optical signal emitted from the sample Sp, which is a photosensitive organism, as image data. And a data processing device 3 that performs drive control of the observation device 5 and performs arithmetic processing using image data sent from the observation device 5, and these two devices cooperate to emit light of the sample Sp. Make observations. In the second embodiment, parts having the same functional configuration as those of the optical signal observation system 1 according to the first embodiment described above are denoted by the same reference numerals.

  The observation device 5 includes a slide glass 51 and a cover glass 52 that house a sample Sp, a movable stage 22, and a light emission image capturing unit that captures a light emission image formed by a light emission image as a signal emitted from the sample Sp. 53, a stage drive unit 24, a transmission illumination unit 54 that irradiates the sample Sp with white light for bright-field images, and an optical path switching drive that performs switching of the optical paths provided in the light-emission image capturing unit 53 and the transmission illumination unit 54, respectively. Unit 26.

  The luminescent image pickup unit 53 is realized using an inverted luminescence microscope, and includes an objective lens 531, an imaging lens 532 that forms an image incident through the objective lens 531, and a luminescent image of the sample Sp. An imaging device 533 that captures a bright-field image, a light source 534 that irradiates the sample Sp with stimulation light, a shutter 535 that switches between transmission and shielding of transmitted light emitted from the light source 534, an objective lens 531, and an imaging lens 532 and a mirror 536 that is detachably disposed between the shutter 535 and the shutter 535.

  The transmitted illumination unit 54 includes a light source 541 such as a halogen lamp that emits light for bright field observation, a shutter 542 that switches between irradiation and non-irradiation of white light, and illumination that condenses white light from the light source 541 on the sample Sp. The lens system 543 is provided, and is disposed on the opposite side of the light emission image capturing unit 53 with respect to the sample Sp. The illumination lens system 543 includes a collector lens 543a and a condenser lens 543b, and performs critical illumination on the sample Sp.

  In the observation device 5, the positions of the light emission image capturing unit 53 and the transmission illumination unit 54 sandwiching the sample Sp are upside down with respect to the observation device 2 described in the first embodiment. Therefore, when the sample Sp is placed on the stage 22, as shown in FIG. 10, the sample Sp to be cultured on the agar medium K is placed on the slide glass 51, and the cover glass 52 is placed on the sample Sp from above. Put it on the flat surface. Thereafter, the cover glass 52 is placed under the slide glass 51 by turning upside down and then placed on the stage 22. Thereby, the luminescence observation of the leaves and stems of the sample Sp can be performed by the inverted luminescence image capturing unit 53.

  When it is desired to observe the root of the sample Sp, a luminescent image or a bright field image may be taken with the sample Sp placed on the petri dish 21 as in FIG.

  According to the second embodiment of the present invention described above, as in the first embodiment, it is possible to stably perform light emission observation at the cell level of a photosensitive organism over a long period of time.

(Embodiment 3)
FIG. 11 is a diagram schematically showing an overall configuration of an optical signal observation system according to Embodiment 3 of the present invention. The optical signal observation system 6 shown in the figure can transmit and receive data between the observation device 7 and the observation device 7 that optically observes the optical signal emitted from the sample Sp, which is a photosensitive organism, as image data. And a data processing device 3 that performs drive control of the observation device 7 and performs arithmetic processing using image data sent from the observation device 7, and these two devices cooperate to emit light of the sample Sp. Make observations. In addition, in this Embodiment 3, about the site | part which has the same function structure as the optical signal observation system 1 which concerns on Embodiment 1 mentioned above, and the optical signal observation system 4 which concerns on Embodiment 2, the same code | symbol is attached | subjected. is there.

  The optical signal observation system 6 includes two light emission image capturing units 71 and 72. Among these, the luminescence image pickup unit 71 is realized by using an upright luminescence microscope, and includes an objective lens 231, an imaging lens 232 that forms an image incident through the objective lens 231, and a sample Sp. An image pickup device 233 for picking up a light emission image and a bright field image, a light source 711 for irradiating the sample Sp with stimulating light and transmitted illumination light, and light to be transmitted among the light emitted from the light source are selectively used A spectral switching unit 712 that switches, and a mirror 236 that is detachably disposed between the objective lens 231, the imaging lens 232, and the spectral switching unit 712 are included.

  The spectral switching unit 712 is configured using a rotating plate, and a filter for stimulation light, a through hole for transmitted illumination, and a light blocking part having a function as a shutter are arranged at substantially equal intervals, and the optical path Rotational driving is performed by the switching drive unit 26. For example, a conventionally known rotary filter can be applied to the rotary plate.

  On the other hand, the luminescent image capturing unit 72 is realized by using an inverted luminescent microscope, and provides stimulating light and transmitted illumination light to the objective lens 531, the imaging lens 532, the imaging device 533, and the sample Sp. A light source 721 to be irradiated, a spectral switching unit 722 that selectively switches light to be transmitted among light emitted from the light source, and an objective lens 531, an imaging lens 532, and a spectral switching unit 722 are detachably disposed. A mirror 536. The configuration of the spectral switching unit 722 is the same as the configuration of the spectral switching unit 712.

  The optical signal observation method according to the third embodiment will be described. In the third embodiment, the optical signal observation system 6 performs the same processing as the optical signal observation method according to the first embodiment. However, in the switching processing to the optical path for stimulating light irradiation in step S2, the optical path switching drive unit 26 sets each spectral light source in a state where the stimulating light filter included in each of the spectral switching units 712 and 722 is located on the optical path. Processing to stop the rotating plate of the switching unit is also performed. Further, in the switching process to the bright-field image optical path in step S4, the optical path switching drive unit 26 sets the spectral illumination units 712 and 722 in the state in which the transmission illumination through holes are located on the optical path. Processing to stop the rotating plate of the switching unit is also performed.

  Note that the light sources 711 and 721 simultaneously perform irradiation with stimulation light and irradiation with light for transmitted illumination for the same period. Thereby, for example, the light that has exited from the light source 711 and reflected by the mirror 236 and passed through the sample Sp and reached the light emitting image capturing unit 72 does not reach the image capturing device 533 because the mirror 536 is on the optical path. Similarly, light that has exited from the light source 721, reflected by the mirror 536, passed through the sample Sp, and reached the luminescent image capturing unit 71 does not reach the imaging device 233 because the mirror 236 is on the optical path. For this reason, it is not necessary to affect the imaging of the light emission image and the bright field image of the imaging devices 233 and 533. Note that the imaging device 233 captures a bright field image with the transmission illumination of the mirror 236, while the imaging device 533 captures the bright field image with the transmission illumination of the mirror 536.

  According to Embodiment 3 of the present invention described above, it is possible to stably perform light emission observation at the cell level of a photosensitive organism for a long period of time, as in Embodiments 1 and 2 described above.

  In addition, according to the third embodiment, observation using an upright microscope and an inverted microscope can be performed, which is suitable when it is desired to observe a photosensitive organism three-dimensionally.

(Other embodiments)
So far, the first to third embodiments have been described in detail as the best mode for carrying out the present invention, but the present invention should not be limited by these embodiments. For example, phosphorescence may be automatically measured for each experiment, and an appropriate interval time T may be set in real time each time.

  In addition, when a plant is used as a sample, the plant grows as it grows. Therefore, if necessary, a plurality of images are taken along the Z direction (optical axis direction), and these images are used to capture the plant. Image processing for reconstructing a three-dimensional image (stereoscopic image) may be performed.

  Furthermore, in the above-described embodiment, the case where the stimulation light is light that promotes the growth of the photosensitive organism has been taken up. However, in general, the stimulation light can affect the biological activity or behavior of the photosensitive organism. Any light can be used. That is, the stimulation light is not necessarily limited to the light that promotes the growth of the photosensitive organism.

  The present invention can also be applied to the case of observing fluorescence emission using a fluorescent protein as an index as a light signal.

  Thus, the present invention can include various embodiments and the like not described herein, and various design changes and the like can be made without departing from the technical idea specified by the claims. It is possible to apply.

It is a figure which shows typically the whole structure of the optical signal observation system which concerns on Embodiment 1 of this invention. It is a block diagram which shows the function structure of the data processor with which the optical signal observation system which concerns on Embodiment 1 of this invention is provided. It is a flowchart which shows the outline | summary of a process of the optical signal observation method concerning Embodiment 1 of this invention. It is a time chart which shows the flow of a time process of the optical signal observation method concerning Embodiment 1 of this invention. It is a figure which shows a bright field image typically. It is a figure which shows a light emission image typically. It is a schematic diagram which shows the example of a display (1st example) of the image which overlapped the bright field image and the light emission image. It is a schematic diagram which shows the example of a display (2nd example) of the image which overlapped the bright field image and the light emission image. It is a figure which shows typically the whole structure of the optical signal observation system which concerns on Embodiment 2 of this invention. It is a schematic diagram which shows the holding method of a sample. It is a figure which shows typically the whole structure of the optical signal observation system which concerns on Embodiment 3 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 4, 6 Optical signal observation system 2, 5, 7 Observation apparatus 3 Data processing apparatus 21 Container 22 Stage 23, 53, 71, 72 Light emission image pick-up unit 24 Stage drive part 25, 54 Transmission illumination unit 26 Optical path switching drive part 31 Transmission / Reception Unit 32 Input Unit 33 Control Unit 34 Storage Unit 35 Output Unit 51 Slide Glass 52 Cover Glass 231, 531 Objective Lens 232, 532 Imaging Lens 233, 533 Imaging Devices 234, 251, 534, 541, 711, 721 Light Source 235 , 252, 535, 542 Shutter 236, 536 Mirror 253, 543 Illumination lens system 253 a, 543 a Collector lens 253 b, 543 b Condenser lens 331 Light emission amount measurement unit 332 Condition determination unit 333 Image generation unit 334 Clock 341 Image data憶部 342 measurement condition storage unit 343 emitting amount storage unit 712, 722 spectral switch unit P1 bright field image P2 luminescent image P3, P4 superimposed image Sp Sample

Claims (9)

Has a light-sensitive, there is provided an optical signal observation method for observing the optical signal plant which is labeled light sensitive organism by photoprotein emits light signals emitted in response to biological activity or behavior,
A stimulation light irradiation step of irradiating the plant body with stimulation light having a component that the plant body senses, based on a predetermined condition;
A light emission image imaging step of imaging the light signal emitted by the plant body irradiated with the stimulation light in the stimulation light irradiation step and imaging a light emission image by imaging with an upright microscope ,
Have
An optical signal observation method comprising providing an interval between the end of the stimulation light irradiation step and the start of the light emission image capturing step. The optical signal observation method according to claim 1, wherein the interval time is determined based on a temporal change of phosphorescence generated around the plant body . The luminescent image correction step of adding a correction based on a temporal change of phosphorescence generated around the plant body to the luminescent image captured in the luminescent image capturing step, according to claim 1 or 2. Optical signal observation method.   The optical signal observation method according to claim 1, wherein the stimulation light irradiation step and the light emission image capturing step are repeatedly performed under a predetermined condition. A bright-field image capturing step of capturing a bright-field image of the plant while irradiating light for bright-field observation after the stimulation light irradiation step and before the interval;
An image generating step for generating an image in which the bright field image captured in the bright field image capturing step and the light emitting image first captured in the light emitting image capturing step after capturing the bright field image are superimposed;
An output step of outputting a superimposed image of the bright-field image and the light-emitting image generated in the image generation step;
The optical signal observation method according to claim 1, further comprising: The stimulation light is red light and / or blue light,
The light signal observation method according to claim 5, wherein the light for bright field observation is white light. The optical signal observation method according to any one of claims 1 to 6 , wherein the photoprotein is luciferase. An optical condition of an imaging optical system including an objective lens and an imaging lens included in the upright microscope, which is expressed by a numerical aperture (NA) and a projection magnification (β) (NA / β) ² The optical signal observation method according to any one of claims 1 to 7, wherein the value of is not less than 0.01. Has a light sensitivity, an optical signal observation system for observing the light signals plant emits a labeled light sensitive organism by photoprotein emits light signals in accordance with the biological activity or behavior,
Stimulation light irradiating means for irradiating the plant body with stimulation light having a component that the plant body senses, based on a predetermined condition;
Is achieved by upright microscope, a luminescent image pickup means for capturing an emission image by focusing light signals the plant emitted,
Control means for performing control to provide an interval between the time when the stimulation light irradiation means ends irradiation of the stimulation light and the time when the light emission image capturing means starts capturing the light emission image;
An optical signal observation system comprising:

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