JP5239472B2 - Fluorescence imaging device - Google Patents

Description

  The present invention relates to an optical bioimaging technique for a biological sample such as a small animal.

  The technique of imaging how molecular species in a living body are distributed is an important research method in medicine and biology. Until now, a method of imaging molecular species using a microscope with a molecular probe (hereinafter referred to as a fluorescent probe) or a chemiluminescent molecular probe using a microscope has been widely used at the cellular level. It was. In the future, there will be a demand for an apparatus for observing the distribution of the molecular species of interest from the cellular level to larger organs and even individual animals. For example, it is a technique in which a fluorescent probe binds to a cancer cell in an individual such as a mouse to image the state of growth of the cancer cell of interest and observe it as a time-dependent change such as daily or weekly. In order to observe the growth of cancer cells inside an animal individual with a conventional measuring device for cell level, the animal is killed and a predetermined part is stained or a fluorescent substance is attached, but then, It is not possible to see the growth of cells over time for a single individual. For this reason, it is desired to develop a device that can observe the molecular species of the internal information of a small animal individual while the individual is alive.

Since near-infrared light has a relatively good light transmittance inside the living body, an apparatus for observing a biological sample uses light having a wavelength of about 600 nm to 900 nm as excitation light for exciting fluorescence.
A conventional fluorescence imaging apparatus for observing such a biological sample can usually only capture an observation image from above (one direction) of the biological sample, and cannot acquire an observation image from multiple directions. When imaging a small animal using a fluorescent probe, fluorescence is not detected when an image is taken from a specific direction, but may be detected when an image is taken from another direction. This means that observation from only one direction may overlook cancer. Therefore, it is necessary for the operator to perform observation from multiple directions approximately by capturing an image obtained by rotating a small animal little by little around the body axis. However, when the biological sample is rotated and images are taken many times, the biological sample is not in a uniform shape. Therefore, it is difficult to perform reproducible measurement because the rotation angle varies each time the biological sample is rotated.

As a method for obtaining observation images from multiple directions, a method has been proposed in which images of a large number of angles are sequentially acquired in a time division manner using a rotating reflector (see Patent Document 1). According to this method, although there is parallel movement of the sample itself, multi-directional observation can be performed by rotating the reflecting mirror and changing the position of the sample without rotating the sample or rotating the two-dimensional detector. It is possible to improve the oversight of fluorescence from the sample by acquiring observation images from multiple directions with high reproducibility.
US Patent Application Publication No. 20050201414

  By the way, the conventional fluorescence imaging apparatus includes only one light source for irradiating excitation light that excites fluorescence, and the relative positional relationship between the irradiation direction of the light source and the observation direction is always constant, In most cases, light is irradiated from substantially the same direction as the observation direction. When the irradiation direction of the light source is the same as the observation direction, autofluorescence generated on the surface of the biological sample is strongly detected and appears as an image in the background. Strong fluorescence can be detected when the position of the fluorescent probe is shallow and close to the side of the biological sample irradiated with the excitation light, but when the probe is deep in the biological sample, Since it attenuates, its detection intensity becomes weak, and it may be buried in the background due to autofluorescence.

  Therefore, when the probe is located deep in the biological sample, it may be better to irradiate the excitation light from a direction different from the observation direction. In addition, there is also an advantage that information in the depth direction of the probe position can be obtained by changing the irradiation direction of the excitation light and performing imaging. In this way, in measurement using biofluorescence imaging technology, it is effective to change the observation direction and excitation light irradiation direction according to conditions such as the depth of the region to be observed and the autofluorescence intensity. is there.

  Accordingly, an object of the present invention is to provide a fluorescence imaging apparatus that can perform imaging under conditions according to the position of a part to be measured on a biological sample.

The present invention includes a sample stage on which a biological sample is placed, an imaging optical system for guiding light of observation images from a plurality of different directions of the biological sample on the sample stage to the two-dimensional detector and the two-dimensional detector. A light source device capable of selectively irradiating excitation light that excites a biological sample on a sample stage and generates fluorescence from two or more directions different from each other as viewed from a two-dimensional detector; A fluorescence filter that shields and guides fluorescence from the biological sample to the two-dimensional detector; an imaging condition setting unit for setting imaging conditions including an observation direction of imaging by the imaging unit and an irradiation direction of excitation light of the light source device; An imaging control unit that controls the light source device and the imaging unit so as to capture an observation image under the imaging conditions set by the imaging condition setting unit, and an image storage unit that stores an image captured by the imaging unit; , Which is stored in the image storage unit An image display unit for displaying at least a portion of the image is a fluorescence imaging apparatus provided with.
Since it has a light source device that can irradiate excitation light from a plurality of directions, an imaging unit that can capture observation images from a plurality of directions, and a condition setting unit that can set the irradiation direction and observation direction of excitation light as imaging conditions, Imaging under arbitrary imaging conditions is possible.

  In a fluorescence imaging apparatus having a plurality of excitation light irradiation directions and a plurality of observation directions, it is possible to acquire captured images of the number of irradiation directions × the number of observation directions for the same biological sample. However, since fluorescence observation with a fluorescence imaging apparatus takes a long time just to acquire one image, it takes an enormous amount of time to acquire all types of images. As in the present invention, if the observation direction and the excitation light irradiation direction can be set as conditions for an image to be captured, it is not always necessary to perform all imaging, and the imaging time of an unnecessary image can be shortened. This function is particularly effective when the position of the site to be observed on the biological sample is known in advance. That is, since it is possible to perform imaging only under conditions suitable for the position of the site to be observed, it is possible to shorten the time required for acquiring the observation image. In addition, what is necessary is just to set so that it may image on all imaging conditions, when the position of the site | part which should observe the biological sample is unknown.

The fluorescence imaging apparatus of the present invention further includes a display image setting unit for setting an image to be displayed in parallel on the image display unit from the image stored in the image storage unit, and the image display unit It is preferable that a function for displaying in parallel the images set by the image condition setting unit among the images stored in the storage unit is provided.
The function of setting images to be displayed in parallel by the image condition setting unit and displaying the set images in parallel on the image display unit is particularly effective when the position of the site to be observed of the biological sample is unknown. As described above, when the position of the part to be observed of the biological sample is unknown, it is necessary to perform imaging in all combinations of the irradiation direction and the observation direction. Depending on the number of observation directions, the amount becomes enormous, and it becomes difficult to view all these images. Thus, if only images used for comparison for obtaining necessary information are selected from the captured images and displayed in parallel, the comparison target image becomes easy to see.

The imaging optical system in the fluorescence imaging apparatus of the present invention is provided at a plurality of different positions around the biological sample on the sample stage, and a plurality of reflecting mirrors that simultaneously guide the light of the observation image from each direction to the two-dimensional detector , and a focusing lens for focusing the light image guided by the plurality of reflecting mirrors on the two-dimensional detector. Therefore , it is possible to simultaneously obtain observation images from a plurality of angles of the biological sample on the sample table by the two-dimensional detector. In that case, the imaging condition setting unit sets only the irradiation direction of the excitation light of the light source device.

  In the above case, the imaging optical system is preferably provided with an auxiliary imaging lens that corrects the focus of the image based on the optical path length difference of the light of each image guided onto the two-dimensional detector by the reflecting mirror.

  In the fluorescence imaging apparatus of the present invention, the light source device includes an illumination light source for capturing an outline image of a biological sample, and illumination light is emitted from the illumination light source to the biological sample when capturing the outline image of the biological sample. In addition, it is preferable that imaging is performed by the imaging unit in a state where the fluorescent filter is out of the optical path of the light of the image from the biological sample. Then, a captured image of a contour image of the biological sample can be acquired, and it is possible to easily confirm from where the fluorescence is emitted by matching the acquired contour image and the fluorescence image.

  In the fluorescence imaging apparatus of the present invention, a light source device capable of irradiating excitation light from a plurality of directions, an imaging unit capable of capturing observation images from a plurality of directions, and the irradiation direction and observation direction of excitation light can be set as imaging conditions. A condition setting unit is provided, so that images can be acquired and displayed in any combination of the irradiation direction and the observation direction, so if the position of the part of the biological sample that you want to observe is known in advance, Imaging can be omitted and imaging can be performed only under imaging conditions corresponding to the position, and the time required for imaging can be shortened. In addition, when the position of the part of the biological sample to be observed is unknown, it is possible to perform imaging in all combinations of the irradiation direction and the observation direction, and selectively display an arbitrary image from the images. Since it can be displayed on the part, a lot of information about the measurement site can be obtained.

  In addition, if any image can be selectively displayed in parallel on the image display unit, a plurality of images to be compared in order for the operator to obtain desired information are displayed in parallel. It becomes easy to do.

An embodiment of the fluorescence imaging apparatus of the present invention will be described below. FIG. 1 is a block diagram schematically showing a configuration of a fluorescence imaging apparatus according to an embodiment.
The biological sample 4 to be observed with this fluorescence imaging apparatus has a site to be observed labeled with a fluorescent agent. The imaging unit 2 for capturing observation images of the biological sample 4 from multiple directions guides light of the image of the biological sample 4 from multiple directions to the two-dimensional detector (not shown) and the two-dimensional detector. Optical system (not shown). The imaging operation of the imaging unit 2 is controlled by the control unit 8.

  There is provided a light source device 6 that irradiates the biological sample 4 with excitation light that excites a portion labeled with a fluorescent agent to generate fluorescence. The light source device 6 can selectively irradiate excitation light from a plurality of different directions. In the light source device 6, the light irradiation operation is controlled by the control unit 8.

  The arithmetic processing unit 9 and the image storage unit 10 are connected to the imaging unit 2. The arithmetic processing unit 9 performs a predetermined process on the image captured by the imaging unit 2 or the image stored in the image storage unit 10 and sends the processed image to the image storage unit 10 or the image display unit 12. The image storage unit 10 can store an image captured by the imaging unit 2 and an image after a predetermined process is performed by the arithmetic processing unit 9. The processing operation of the arithmetic processing unit 9 is controlled by the control unit 8.

The control unit 8 controls the imaging unit 2, the light source device 6, and the arithmetic processing unit 9. The control is performed based on information set by the imaging condition setting unit 14 and the display image setting unit 16.
In the imaging condition setting unit 14, an observation direction for imaging and an irradiation direction of excitation light are set by the operator. In the display image setting unit 16, an image to be displayed on the image display unit 12 is set by an operator. The image display unit 12 has a function of displaying not only the images picked up by the image pickup unit 2 in a list but also displaying the images set by the display image setting unit 16 in parallel.

Next, a specific example of the configuration of the imaging unit 2 and the light source device 6 of the fluorescence imaging apparatus of the embodiment will be described with reference to FIG.
The biological sample 4 is placed on a transparent sample table 3. As the imaging unit 2, a two-dimensional detector 18, a reflecting mirror M1 to M4, a main imaging lens 20, an auxiliary imaging lens L0 to L4, and a fluorescence filter 22 are provided immediately above the sample stage 3.

  The reflecting mirrors M <b> 1 to M <b> 4 are arranged in a plane perpendicular to the main plane direction of the sample stage 3. The reflecting mirrors M1 to M4 set the direct observation direction from directly above by the two-dimensional detector 18 to 0 °, and the light of the observation image from the direction shifted every 72 ° counterclockwise to the two-dimensional detector 18. Arranged to guide. That is, the reflecting mirror M1 is arranged to direct the light of the observation image from the 72 ° direction, the reflecting mirror M2 to the 144 ° direction, the reflecting mirror M3 to the 216 ° direction, and the reflecting mirror M4 to the two-dimensional detector 18 from the 288 ° direction. Has been.

The main imaging lens 20 forms an image of the light from the reflecting mirrors M1 to M4 on the two-dimensional detector 18, and is provided on the light receiving surface side of the two-dimensional detector 18.
The auxiliary imaging lenses L0 to L4 are for correcting the optical path length difference of the light from the respective reflecting mirrors M1 to M4, and are disposed on the respective optical paths.
The fluorescence filter 22 shields the excitation light and transmits the fluorescence component, and is provided on the incident side of the main imaging lens 20 when observing fluorescence from the biological sample 4.

  The light source device 6 includes five light sources S1 to S5. The light sources S <b> 1 to S <b> 5 of the light source device 6 are provided in a plane perpendicular to the body axis of the biological sample 4 and are equally arranged at different positions on the same circumference centering on the body axis extension line of the biological sample 4. Has been. The light sources S <b> 1 to S <b> 5 are 36 ° (S 1), 108 ° (S 2), 180 ° (S 3), 252 ° (S 4), 324 counterclockwise with the direction from right above the biological sample 4 as 0 ° direction. It arrange | positions so that light may be irradiated from the direction of (S5). These light sources S1 to S5 can be turned on and off independently. In the drawing, the light source device 6 shows only the circumference that holds the light sources S1 to S5. In addition, although the light sources S1 to S5 are illustrated as one light source in the figure, these light sources S1 to S5 are excitations that excite specific parts of the biological sample 4 labeled with a fluorescent agent to generate fluorescence. An excitation light source that emits light and an illumination light source for capturing an entire image of the biological sample 4 are provided.

  In the fluorescence imaging apparatus of this example, the light of the direct observation image by the two-dimensional detector 18 and the light of the observation image guided by the reflecting mirrors M1 to M4 are connected on the two-dimensional detector 18 by the main imaging lens 20. And observation images from five directions as shown in FIG. 3 and FIG. 4 (A) can be simultaneously acquired. FIG. 4 (A) is an image obtained by superimposing a fluorescence observation image on an externally captured external image. For example, as shown in FIG. 4B, the fluorescence observation image captured in this way is cropped for each observation direction so as to be easily confirmed by the operator, and is horizontally reversed. Processing such as adjustment and display order adjustment is performed and displayed on the image display unit 12.

  In the configuration of FIG. 2, observation images from five directions are formed on the two-dimensional detector 18 and simultaneously imaged. Therefore, the imaging condition set by the imaging condition setting unit 14 is the excitation light irradiation direction (S1 to S5). ) Only. As a setting method of the imaging condition in this case, a method of selecting and setting a sequence number from the sequence list shown in Table 1 can be cited. In Table 1, “◯” means that the light source is turned on, and “x” means that the light source is turned off.

The example in Table 1 includes a step (sequence 1) of lighting all the light sources S1 to S5 of the light source device 6 and a step of shooting (sequence 2 to sequence 6) of lighting each of the light sources S1 to S5 one by one. The case where it provided is shown. A list of such sequences is set in the imaging condition setting unit 14, and the imaging condition setting unit 14 is changed to the selected sequence when the operator selects a sequence corresponding to the position of the part to be observed of the biological sample 4. A corresponding signal is transmitted to the control unit 8, and the control unit 8 controls the imaging unit 2 and the light source device 6 so that imaging is performed under conditions according to the selected sequence.
In this embodiment, observation images from five directions can be taken simultaneously for one sequence (irradiation direction of excitation light). Therefore, for example, when all the sequences 1 to 6 are executed, 6 × 5 = 30 types of observations are performed. Images can be acquired. Table 2 shows an example of assignment of image numbers to observation images obtained by executing all sequences.

  In the example of Table 2, numbers 1 to 5 are assigned to the five types of images acquired in the sequences 1 to 6 in the order of the observation direction. The images allocated here were cut out for each observation direction from five types of images captured as one image in one sequence, and adjusted so that the operator could easily confirm such as left-right reversal and size adjustment. It is an image. By listing in this way, the operator can take out and display only the images to be confirmed.

FIG. 5 shows an example in which images selected by the operator are displayed in parallel on the image display unit 12. The images displayed in parallel on the image display unit 12 correspond to the display image conditions input by the operator to the display image condition setting unit 16 as shown in Table 3, for example. In this example, the case where the operator inputs five image conditions is shown. When four or less or six or more image conditions are specified, an image corresponding to the conditions is displayed on the image display unit 12. Further, as shown in FIG. 6, a plurality of sets of images to be displayed in parallel on the image display unit 12 may be set.

  In the fluorescence imaging apparatus of this embodiment, as shown in FIGS. 2 and 3, five images can be acquired simultaneously in one sequence, but these images were taken from different observation directions in the same excitation light irradiation direction. It is an image. However, if images arbitrarily selected in this way can be displayed in parallel, for example, images having the same observation direction but different excitation light irradiation directions can be displayed and compared in parallel.

In Table 1, a sequence (sequence 1) for turning on all the light sources S1 to S5 of the light source device 6 and a sequence (sequence 2 to sequence 6) for turning on only one of the light sources are provided. As shown in FIG. 4, a sequence for simultaneously lighting two or more light sources may be further provided.

Next, the operation of the fluorescence imaging apparatus of this embodiment will be described with reference to FIG. 1, FIG. 7 and FIG. FIG. 7 is a flowchart illustrating an example of an imaging operation of the fluorescence imaging apparatus, and FIG. 8 is a flowchart illustrating an example of an operation for displaying a captured image.
First, the imaging condition setting unit 14 sets imaging conditions according to the position of the part that the operator wants to observe (step S1). Details of the imaging condition setting method will be described later.

  In a state in which the biological sample 4 is irradiated with illumination light (not excitation light), an entire image from all the observation directions of the biological sample 4 as shown in FIG. 3 is captured (step S2). At this time, the fluorescence filter 22 for guiding only the fluorescence component to the two-dimensional detector 18 as shown in FIG. 2 is removed from the optical path of the light from each observation direction. The captured image of the whole image of the biological sample 4 is stored in the image storage unit 10 (step S3). In addition, when an observation image from a plurality of directions is captured as a single image, the image captured by the arithmetic processing unit 9 is divided for each observation direction, corrected for size adjustment, left / right inversion, and the like, and then stored in an image. You may make it memorize | store in the part 10. FIG.

Next, a fluorescence observation image of the biological sample 4 is captured based on the imaging conditions set by the imaging condition setting unit 14. In this imaging, the lighting of the light source of the light source device 6 and the optical system of the imaging unit 2 are adjusted so that the imaging can be performed in the excitation light irradiation direction and the observation direction set by the imaging condition setting unit 14 (step S4). The image is taken (step S5). The captured fluorescence observation image is stored in the image storage unit 10 (step S6). In addition, when an observation image from a plurality of directions is captured as a single image, the image captured by the arithmetic processing unit 9 is divided for each observation direction, corrected for size adjustment, left / right inversion, and the like, and then stored in an image. You may make it memorize | store in the part 10. FIG.
When a plurality of imaging conditions are set in the imaging condition setting unit 14, adjustment of the irradiation direction and observation direction of the excitation light (step S4) and imaging (step S5) until imaging under all imaging conditions is completed. The captured image is repeatedly stored (step S6) (step S7).

  After imaging under all imaging conditions is completed, the operator selects whether to display the captured image on the image display unit 12 (step S11). When displaying an image on the image display unit 12, the operator can further select whether or not to display a specific image in parallel (step S12). When displaying a specific image in parallel on the image display unit 12, the operator selects and sets an image to be displayed in parallel in the display image setting unit 16 (step S13), and the set image is displayed in parallel on the image display unit 12. (Step S14). When specific images are not displayed in parallel, all captured images are displayed in a list (step S15). A method for setting images to be displayed in parallel will be described later.

  In the above operation, the fluorescent image is captured (steps S4 to S6) after the entire image of the biological sample 4 is captured (step S2), but actually, the entire image of the biological sample 4 is captured. Imaging may be performed after imaging of the fluorescent image.

An imaging condition setting method will be described with reference to FIG. FIG. 9 is a flowchart illustrating an example of the imaging condition setting method.
In setting the imaging conditions, the operator selects the excitation light irradiation direction (step S21), and selects the observation direction to be imaged when the excitation light is irradiated from the irradiation direction (step S22). When the imaging unit 2 sequentially captures observation images from a plurality of directions, for example, the imaging unit 2 includes a rotating reflector, the observation direction can be selected. In the case where 2 is to simultaneously capture observation images from a plurality of directions as shown in FIG. 2, no observation direction is designated. The combination of the excitation light irradiation direction and the observation direction thus set is stored as an imaging condition (step S23). When further imaging conditions are set (step S24), the irradiation direction of the excitation light is selected again (step S21), and the observation direction desired to be imaged in the irradiation direction is selected (step S22). Stored (step S23). The set imaging conditions may be stored in the imaging condition setting unit 14 or may be stored in the control unit 8.

Next, an example of a setting method for images to be displayed in parallel on the image display unit 12 will be described. FIG. 10 is a flowchart illustrating an example of a method for setting an image to be displayed in parallel on the image display unit 12.
First, the operator sets the number of images to be displayed in parallel on the image display unit 12 (step S31). By setting the number of images to be displayed in parallel by the operator, the arrangement position of the display image is assigned and the arrangement position can be designated. The operator selects an image to be displayed in parallel from the images stored in the image storage unit 10 (step S32), and the position where the image is to be arranged in the image display unit 12 is determined. Select (step S33). The selected image and the arrangement position of the image are stored in the display image setting unit 16 or the control unit 8 (step S34), and the above operations are repeated until images to be displayed at all the arrangement positions are selected (step S34). S35).

  In addition, when selecting an image, it is preferable that the images stored in the image storage unit 10 are displayed in a list together with the imaging conditions in the image display unit 12 or can be searched and selected from the imaging conditions. . The operator may be able to select whether or not image processing is performed by the arithmetic processing unit 9 on the selected image.

  The observation results when the fluorescence observation is actually performed using the fluorescence imaging apparatus of FIG. 2 are shown below. In this observation, a phantom 24 shown in FIG. The phantom 24 is made of a scattering material and a resin mixed with the absorbing material so that light is scattered and absorbed and propagated like a biological sample, and is cured into a cylindrical shape having a diameter of 28 mm. Fluorescent agents a and b that emit fluorescence when irradiated with excitation light at two different locations in the phantom 24 are installed. The fluorescent agent a is at a position 2 mm from the surface, and the depth of the fluorescent agent b is at a position 4 mm from the surface.

  In this fluorescence observation, FIGS. 12 to 16 show images taken simultaneously from five directions when the light sources S1 to S5 of the light source device 6 are turned on one by one in order. 12 shows only the light source S1 (36 °), FIG. 13 shows only the light source S2 (108 °), FIG. 14 shows only the light source S3 (180 °), FIG. 15 shows only the light source S4 (252 °), FIG. Is an observation image when only the light source S5 (324 °) is turned on and imaged.

  As can be seen from a comparison of FIGS. 12 to 16, when the fluorescent agent is present in a shallow portion of the sample, stronger fluorescence can be detected by observing from a direction closer to the fluorescent agent. Is located far from the irradiation direction of the excitation light, strong fluorescence cannot be detected even when observed from a direction close to the fluorescent agent. Further, as the position of the fluorescent agent approaches the center of the sample, the dependence of the fluorescence detection intensity on the excitation light irradiation direction decreases. That is, if an image that is captured so that the operator can easily confirm not only the dependency of the fluorescence detection intensity on the observation direction but also the dependency of the fluorescence detection intensity on the excitation light irradiation direction is displayed, a more detailed description of the fluorescent agent can be obtained. Location information can be obtained.

  FIG. 17 is a diagram in which images with different excitation light irradiation directions in the same observation direction are cut out from a total of 25 types of images captured in FIGS. 12 to 16 and arranged in parallel. (A) is an image in which observation images from an observation direction of 0 ° in which the fluorescence intensity from the fluorescent agent a of the phantom 24 is detected are arranged for each irradiation direction of the excitation light, and (B) is from the fluorescent agent b. The observation images from the observation direction of 288 ° where the fluorescence intensity is detected are arranged for each excitation light irradiation direction. When these image data are compared, in (B), the fluorescence from the fluorescent agent b is strongly detected in the excitation light irradiation directions 252 ° and 324 ° which are close to the observation direction 288 °. Almost no detection. On the other hand, in (A), the fluorescence is strongly detected in the excitation light irradiation directions of 36 ° and 324 ° at the observation direction of 0 °, and the fluorescence from the fluorescent agent a is weak in the images of the irradiation directions of 108 ° and 252 °. Fluorescence is detected. This gives the knowledge that the fluorescent agent a is deeper than the fluorescent agent b.

  FIG. 18 is a diagram in which images having a relative positional relationship of 36 ° (± 36 °) between the observation direction and the excitation light irradiation direction are extracted from the images of FIGS. 12 to 16 and arranged. . In other words, these images simulate images from multiple observation directions obtained using a conventional reflecting mirror rotation type fluorescence imaging apparatus in which the relative positional relationship between the observation direction and the excitation light irradiation direction is always the same. It is shown as an example.

  From the five images shown in FIG. 18, it is estimated that there are two fluorescent agents in the phantom 24, and it is understood that there is no oversight that occurs in unidirectional irradiation / unidirectional observation. In this method, since imaging is performed by irradiating excitation light from a direction that is always close to the observation direction, fluorescence as strong as possible can be detected from an image captured from any observation direction. Therefore, this reflecting mirror rotation type may be used from the viewpoint of reliably detecting fluorescence whose location is unknown. However, if it is desired to detect fluorescence as strongly as possible from any observation direction, the same result can be obtained by simultaneously irradiating excitation light from all directions and imaging using a multi-directional simultaneous observation apparatus as shown in FIG. Can be obtained. Furthermore, since fluorescence observation for a living body is basically faint light observation, it takes time to acquire one image using a storage type two-dimensional detector such as a CCD camera. A reflection mirror rotation type fluorescence imaging apparatus requires a great deal of time to acquire all images, and an apparatus capable of acquiring observation images from multiple directions at the same time requires a shorter time to acquire all images.

  Further, for example, when fluorescence from the fluorescent agent b is detected stronger than the fluorescent agent a, it indicates that the fluorescent agent b has a higher concentration than a or is located at a position shallower than the fluorescent agent a. Cannot get information to confirm what is shown. In this rotating mirror type, the relative positional relationship between the irradiation direction and the observation direction is always fixed, so it is not possible to acquire images when the excitation light is irradiated from different directions in the same observation direction. is there.

  In addition, when the fluorescent agent is present near the center of the biological sample, the dependency of the detected fluorescence intensity on the irradiation direction and the dependency on the observation direction is extremely reduced, and the intensity is absorbed by the living body. Become extremely weak. In this case, when the irradiation direction is irradiated from the same side as the observation, the intensity of autofluorescence generated on the surface of the biological sample is also observed strongly, so that the possibility of being buried in the background of autofluorescence increases. On the other hand, in multi-directional irradiation / multi-directional observation, since a biological sample can be observed from the irradiation direction on the opposite side, measurement that is relatively less susceptible to autofluorescence can be performed.

  While the embodiments of the fluorescence imaging apparatus of the present invention have been described above, the functions of the fluorescence imaging apparatus are summarized below.

By arbitrarily setting the imaging conditions, the following is possible.
(1) When the position of the measurement site (fluorescent agent) is known in advance by measurement or the like, and when the change over time of the measurement site is continuously measured, the conditions under which fluorescence can be detected most strongly That is, imaging is performed by setting the observation direction and the irradiation direction from the direction close to the measurement site.
(2) In order to reliably detect a measurement site whose location is unknown without overlooking it, an observation image from all directions is captured in a state where excitation light is irradiated from all directions.
(3) Imaging is performed for all combinations of the irradiation direction and the observation direction in order to acquire information on the depth direction of the measurement site whose location is unknown.

Furthermore, since images to be displayed in parallel on the image display unit 12 can be set, parallel display according to the following purposes is possible.
(1) In order to confirm a measurement site (fluorescence) whose location is unknown, images in which the excitation light irradiation direction and the observation direction are close are displayed in parallel.
(2) In order to obtain depth information on the focused fluorescence, observation images in different irradiation directions in the same observation direction are displayed in parallel.
(3) In order to confirm the fluorescence in the deep position of the biological sample, the observation image from the side opposite to the excitation light irradiation direction is displayed in parallel, that is, the condition with less background due to autofluorescence.

  By the way, in fluorescence measurement using a fluorescence imaging apparatus, it is common to select an optical filter (fluorescence filter) of a selection wavelength of a fluorescence excitation light source or an observation wavelength band. In the embodiment described above, a combination of two elements of the observation direction and the excitation light irradiation direction is set as the imaging condition and the display image condition, but the wavelength of the excitation light and the transmission wavelength band of the fluorescent filter are set. May also be added as a selection element of the imaging condition and the display image condition.

It is a block diagram which shows roughly one Example of a fluorescence imaging apparatus. It is a block diagram which shows specifically the structure of the Example. It is an image which shows an example of the image of the biological sample imaged by one imaging operation. It is an example of a fluorescence observation image, (A) is a fluorescence observation image from five directions imaged simultaneously by one imaging operation, and (B) is a preferred arrangement example of the fluorescence observation image. It is a figure which shows an example of the screen display of an image display part. It is a figure which shows the other example of the screen display of an image display part. It is a flowchart figure for demonstrating an example of the imaging operation of a fluorescence imaging device. It is a flowchart figure for demonstrating an example of the screen display operation | movement of a fluorescence imaging device. It is a flowchart for demonstrating an example of the setting method of an imaging condition. It is a flowchart for demonstrating an example of the setting method of a display image. It is a figure which shows the phantom used in the measurement experiment of a fluorescence image, (A) is a perspective view, (B) is a front view, (C) is a side view. It is the image which imaged the phantom simultaneously from 5 directions in the state which irradiated the excitation light from the direction of 36 degrees. It is the image which imaged the phantom simultaneously from 5 directions in the state which irradiated the excitation light from the direction of 108 degrees. It is the image which imaged the phantom simultaneously from five directions in the state which irradiated the excitation light from the direction of 180 degrees. It is the image which imaged the phantom simultaneously from 5 directions in the state which irradiated the excitation light from the direction of 252 degrees. It is the image which imaged the phantom simultaneously from five directions in the state which irradiated the excitation light from the direction of 324 degrees. FIGS. 12 to 16 are images in which the images in the same observation direction are arranged in the order of the excitation light irradiation direction, (A) is an image in which the observation direction is 0 °, and (B) is an image in which the observation direction is 288 °. 17 is an image in which only images having a relative angle difference of 36 ° between the observation direction and the excitation light irradiation direction are arranged from the images of FIGS.

Explanation of symbols

DESCRIPTION OF SYMBOLS 2 Imaging part 3 Sample stand 4 Biological sample 6 Light source device 8 Control part 9 Arithmetic processing part 10 Image storage part 12 Image display part 14 Imaging condition setting part 16 Display image setting part 18 Two-dimensional detector 20 Main imaging lens 22 Fluorescence filter L0 to L4 Auxiliary imaging lens M1 to M4 Reflector S1 to S5 Light source 24 Phantom

Claims (4)

A sample stage on which a biological sample is placed;
An imaging unit including a two-dimensional detector and an imaging optical system for guiding light of an observation image from a plurality of different directions of the biological sample on the sample table to the two-dimensional detector;
A light source device capable of selectively irradiating excitation light that excites a biological sample on the sample stage and generates fluorescence from two or more directions different from each other as viewed from the two-dimensional detector;
A fluorescence filter that shields the excitation light and guides fluorescence from a biological sample to the two-dimensional detector;
An imaging condition setting unit for setting an imaging condition including an observation direction of imaging by the imaging unit and an irradiation direction of excitation light of the light source device;
An imaging control unit that controls the light source device and the imaging unit so that an observation image under the imaging conditions set by the imaging condition setting unit is captured by the imaging unit;
An image storage unit for storing an image captured by the imaging unit;
An image display unit that displays at least a part of the plurality of images stored in the image storage unit ,
The imaging optical system is provided at a plurality of different positions around the biological sample on the sample stage, and a plurality of reflecting mirrors that simultaneously guide light of an observation image from each direction to the two-dimensional detector; An image forming lens that forms an image of the light of the image guided by the reflecting mirror on the two-dimensional detector,
The fluorescence imaging apparatus which sets only the irradiation direction of the excitation light of the said light source device in the said imaging condition setting part . A display image setting unit for setting an image to be displayed in parallel on the image display unit from among images stored in the image storage unit,
The fluorescence imaging apparatus according to claim 1, wherein the image display unit has a function of displaying in parallel the images set by the display image setting unit among the images stored in the image storage unit. Wherein the imaging optical system, in claim 1 in which the auxiliary image forming lens for correcting the size of the image based on the optical path length difference of light of each image to be directed onto the two-dimensional detector by the reflector is provided The fluorescence imaging apparatus described. The light source device includes an illumination light source for capturing an outline image of a biological sample,
When capturing an external image of a biological sample, illumination light is irradiated from the illumination light source to the biological sample, and imaging is performed by the imaging unit in a state where the fluorescent filter is out of the optical path of the light of the image from the biological sample. The fluorescence imaging apparatus according to any one of claims 1 to 3 , which is performed.