JP2010044018A - Observation apparatus, observation container, and slide glass - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stripe illumination/observation apparatus, an observation container and a slide glass for improving the flexibility of a layout around a to-be-observed object. <P>SOLUTION: An embodiment of the observation apparatus is an observation apparatus for observing the to-be-observed object (15) transparent to a light in a particular wavelength range and includes a fluorescent member (16) repetitively provided with first fluorescent sections at an interval which emit a fluorescence within the particular wavelength range in response to a light having a first wavelength; an illumination optical system (27) for illuminating the fluorescent member with the light having the first wavelength; and an observation optical system (24) for generating an image of a sample (15) illuminated by the fluorescence emitted by the fluorescent member. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an observation apparatus, an observation container, and a slide glass suitable for observing a transparent observation object with a wide field of view.

The observation apparatus described in Patent Document 1 generates a striped dark field image by illuminating an object to be observed with illumination (stripe illumination) in which a bright part and a dark part are repeatedly arranged, and a transparent object to be observed It is possible to observe a wide field of view. In order to realize this striped illumination, it is necessary to arrange a large-scale device such as a liquid crystal display panel near the object to be observed.
JP 2007-178426 A

  However, since the object to be observed may take various forms such as a preparation, a dish, a flask, etc., it is desirable to secure as much space as possible around the object. Further, if the device is arranged near the object to be observed, the state of the object to be observed (particularly, a living cell) may not be kept good due to heat generated by the device.

  Accordingly, an object of the present invention is to provide a stripe illumination observation apparatus, an observation container, and a slide glass that can increase the degree of freedom of layout around an object to be observed.

  One aspect of an observation apparatus illustrating the present invention is an observation apparatus that observes an object to be observed that is transparent to light in a predetermined wavelength range, and emits fluorescence in the predetermined wavelength range in response to light of a first wavelength. A fluorescent member in which first fluorescent portions are repeatedly provided at intervals; an illumination optical system that illuminates the fluorescent member with light of the first wavelength; and an object that is illuminated with fluorescence emitted from the fluorescent member. And an observation optical system for generating an image.

  An aspect of the observation container exemplifying the present invention is an observation container that is transparent to light in a predetermined wavelength range, and the bottom of the observation container is within the predetermined wavelength range according to light of the first wavelength. The first fluorescent part that emits the fluorescence is repeatedly provided at intervals.

  Moreover, one aspect of the slide glass illustrating the present invention is a slide glass that is transparent with respect to a wavelength within a predetermined wavelength range, and emits fluorescence within the predetermined wavelength range in response to light of the first wavelength. Is repeatedly provided at intervals.

  ADVANTAGE OF THE INVENTION According to this invention, the stripe illumination observation apparatus, observation container, and slide glass which can raise the freedom degree of the layout of a to-be-observed object periphery are implement | achieved.

[First Embodiment]
Hereinafter, embodiments of the observation apparatus will be described.

  FIG. 1 is a configuration diagram of an optical system portion of the observation apparatus of the present embodiment. As shown in FIG. 1, the observation apparatus of this embodiment includes a light source 26, an illumination optical system 27, an electric turret 28, an electric stage 22, an imaging optical system 24, an electric turret 29, an image sensor 25, and the like. .

  The stage 22 is a stage provided with a hollow portion, and the transparent fluorescent substrate 16 is loaded in the hollow portion. The transparent fluorescent substrate 16 is a transparent substrate in which a fluorescent layer 16B is formed over substantially the entire lower surface. Details of the transparent fluorescent substrate 16 will be described later.

  An object to be observed 15 that is transparent to at least visible light is placed at substantially the center of the upper surface of the transparent fluorescent substrate 16. Here, the observation object 15 is a dish-shaped culture container (dish) containing a culture solution containing living cells.

  The light source 26 is a white light source such as an LED or a halogen lamp, and the emitted light includes at least light having the same wavelength as the excitation wavelength (described later) of the fluorescent layer 16B.

  The illumination optical system 27 enlarges the diameter of the light beam emitted from the light source 26 and collectively illuminates substantially the entire lower surface of the transparent fluorescent substrate 16.

  The turret 28 is disposed at any location on the optical path from the light source 26 to the transparent fluorescent substrate 16, and is attached with a first excitation filter 28-1 and a second excitation filter 28-2 having different passbands. Yes. When the turret 28 is driven, the excitation filter inserted in the optical path is switched between the first excitation filter 28-1 and the second excitation filter 28-2, so that the light that illuminates the transparent fluorescent substrate 16 (hereinafter referred to as “light source”). The wavelength of “excitation light” is switched between two types of wavelengths.

  The imaging optical system 24 guides only a light beam having a spread angle equal to or smaller than a predetermined value out of each light beam emitted from each part of the observation object 15 to the imaging surface of the imaging device 25. An image of the object to be observed 15 is formed. The field-of-view size of the imaging optical system 24 is sufficiently large. Here, the size is set such that the entire image of the observation object 15 can be captured.

  The image pickup device 25 is a color image pickup device having sensitivity in the visible light range, and generates an image signal by photoelectrically converting the entire image of the observation object 15 formed on the image pickup surface.

  The turret 29 is disposed in any part of the optical path from the object to be observed 15 to the image sensor 25, and is attached with a first barrier filter 29-1 and a second barrier filter 29-2 having different cut bands. ing. When the turret 29 is driven, the barrier filter inserted in the optical path is switched between the first barrier filter 29-1 and the second barrier filter 29-2, and thus light that is cut before the image sensor 25 ( Hereinafter, the wavelength of “barrier light” is switched between two types of wavelengths.

  Here, the first barrier filter 29-1 is a barrier filter to be used together with the first excitation filter 28-1, and the cut band is the same as the pass band of the first excitation filter 28-1. is there. The second barrier filter 29-2 is a barrier filter to be used together with the second excitation filter 29-2 described above, and its cut band is the same as the pass band of the second excitation filter 28-2. .

  FIG. 2 is a configuration diagram of a circuit portion of the observation apparatus of the present embodiment. As shown in FIG. 2, the imaging apparatus driver 41, the barrier filter driver 43, the stage driver 23, the excitation filter driver 40, the light source driver 42, the controller 36, the computer 37, the monitor 54, the input device 55, etc. are included in the observation device of this embodiment. Is provided.

  The image sensor driver 41 drives the above-described image sensor 25 to capture an image signal from the image sensor 25 and sends it to the controller 36 as image data of the entire image of the object 15 to be observed.

  The barrier filter driver 43 switches the wavelength of the barrier light described above between two types of wavelengths by driving the turret 29 described above.

  The stage driver 23 adjusts the distance between the object to be observed 15 and the imaging optical system 24 by driving the stage 22 described above in the optical axis direction.

  The excitation filter driver 40 switches the wavelength of the excitation light described above between two types of wavelengths by driving the turret 28 described above.

  The light source driver 42 turns on / off the excitation light by turning on / off the light source 26 described above.

  The controller 36 controls the image sensor driver 41, the barrier filter driver 43, the stage driver 23, the excitation filter driver 40, and the light source driver 42.

  For example, the controller 36 interlocks the excitation filter driver 40 and the barrier filter driver 43 to change the combination of the excitation filter and the barrier filter inserted into the optical path to the first excitation filter 28-1 and the first barrier filter 29-1. The combination is switched between the combination of the second excitation filter 28-2 and the second barrier filter 29-2.

  Further, the controller 36 acquires the image data of the object to be observed 15 by controlling the timing at which the light source driver 42 drives the light source 26 and the timing at which the image sensor driver 41 drives the image sensor 25, and transmits it to the computer 37. To do.

  The computer 37 performs image processing on the image data transmitted from the controller 36, converts the image data after the image processing into image data for display, and outputs the image data to the monitor 54. Therefore, the user can observe the entire image of the observation object 15 on the monitor 54.

  Further, the computer 37 displays various GUI images on the monitor 54. The user is prompted by the GUI image and operates the input device 55 to input various instructions related to observation to the computer 37. This instruction is transmitted from the computer 37 to the controller 36 as necessary, and control of each unit by the controller 36 is performed according to the instruction.

  For example, if the user inputs an instruction to move the stage 22 up and down, the instruction is transmitted from the computer 37 to the controller 36, and the controller 36 causes the stage driver 23 to move the stage 22 up and down according to the instruction and The distance between the object 15 and the imaging optical system 24 is adjusted. Accordingly, it is possible to adjust the focus of the imaging optical system 24 with respect to the object 15 to be observed.

  Here, it is assumed that the focus adjustment is performed by driving the stage 22, but a mechanism for moving all or part of the lens of the imaging optical system 24 is provided, and the focus adjustment is performed by driving the mechanism. May be. Hereinafter, it is assumed that the focal point of the imaging optical system 24 is set on the bottom of the object to be observed (here, dish) 15.

  FIG. 3 is a diagram for explaining the transparent fluorescent substrate 16 of this embodiment in detail. FIG. 3A is a cross-sectional view obtained by cutting the transparent fluorescent substrate 16 along a plane parallel to the optical axis. FIG. 3B shows the transparent fluorescent substrate 16 as viewed from the illumination optical system 27 side. FIG.

  A main body (substrate main body) 16A of the transparent fluorescent substrate 16 is a parallel flat plate (for example, a glass substrate) transparent to visible light. The fluorescent layer 16B formed on the lower surface of the substrate body 16A is a layer obtained by mixing a fluorescent material with a material transparent to visible light (for example, a thermosetting resin). The fluorescent layer 16B may be a layer formed by applying a fluorescent paint on the substrate body 16A.

  In the fluorescent layer 16B, strip-shaped first fluorescent regions 16B-1 and strip-shaped second fluorescent regions 16B-2 are alternately arranged without gaps in a predetermined direction (left-right direction in FIG. 3) perpendicular to the optical axis. . The width of the first fluorescent region 16B-1 and the width of the second fluorescent region 16B-2 are equal, and the arrangement pitch of the first fluorescent region 16B-1 and the second fluorescent region 16B-2 is uniform.

  The characteristics of the first fluorescent region 16B-1 (that is, the characteristics of the fluorescent substance included in the first fluorescent region 16B-1) and the characteristics of the second fluorescent region 16B-2 (that is, the fluorescence included in the second fluorescent region 16B-2) The characteristics of the first excitation filter 28-1, the second excitation filter 28-2, the first barrier filter 29-1, and the second barrier filter 29-2 are different from each other in accordance with these characteristics. Is set.

  FIG. 4 is a diagram showing the relationship between the characteristics of the first fluorescent region 16B-1 and the characteristics of the first excitation filter 28-1. In FIG. 4, the reference symbol Sa-1 indicates the absorption spectrum of the first fluorescent region 16B-1, and the reference symbol Se-1 indicates the emission spectrum of the first fluorescent region 16B-1, denoted by reference symbol T1. A wavelength characteristic curve of the transmittance of the first excitation filter 28-1 is shown. Although not shown in the figure, the wavelength characteristic curve of the transmittance of the first barrier filter 29-1 corresponds to an inverted version of the wavelength characteristic curve of the transmittance of the first excitation filter 28-1.

  As shown in FIG. 4, the excitation wavelength of the first fluorescence region 16B-1 is blue, and the fluorescence wavelength (first fluorescence wavelength) of the first fluorescence region 16B-1 is green. The pass band of the first excitation filter 28-1 is set to the same wavelength (blue) as the excitation wavelength of the first fluorescent region 16B-1, and the cut band of the first barrier filter 29-1 is the same as that. The wavelength (blue) is set.

  FIG. 5 is a diagram illustrating the relationship between the characteristics of the second fluorescent region 16B-2 and the characteristics of the second excitation filter 29-2. In FIG. 5, the reference symbol Sa-2 indicates the absorption spectrum of the second fluorescent region 16B-2, and the reference symbol Se-2 indicates the emission spectrum of the second fluorescent region 16B-2. Shown is the wavelength characteristic curve of the transmittance of the second excitation filter 28-2. Although not shown in the figure, the wavelength characteristic curve of the transmittance of the second barrier filter 29-2 corresponds to an inverted version of the wavelength characteristic curve of the transmittance of the second excitation filter 28-2.

  As shown in FIG. 5, the excitation wavelength of the second fluorescent region 16B-2 is green, and the fluorescent wavelength of the second fluorescent region 16B-2 is red. The pass band of the second excitation filter 28-2 is set to the same wavelength (green) as the excitation wavelength of the second fluorescent region 16B-2, and the cut band of the second barrier filter 29-2 is the same as that. Wavelength (green) is set.

  Therefore, in the observation apparatus of this embodiment, when the combination of the excitation filter and the barrier filter inserted in the optical path is switched, the wavelengths of the excitation light and the barrier light are the same as the excitation wavelength of the first fluorescent region 16B-1 (blue). ) And the same wavelength (green) as the excitation wavelength of the second fluorescent region 16B-2.

  FIG. 6 is a schematic diagram showing a state around the object 15 when the wavelength of the excitation light (and the barrier light) is the same as the excitation wavelength (blue) of the first fluorescent region 16B-1. In FIG. 6, the symbol Lb indicates excitation light (blue light), and the symbol Lg indicates fluorescence (green light).

  As shown in FIG. 6, when the wavelength of the excitation light (and the barrier light) is the same as the excitation wavelength (blue) of the first fluorescent region 16B-1, the first fluorescent region 16B-1 is excited and the second fluorescent region 16B. -2 is not excited. Therefore, fluorescence (green light) is generated in the first fluorescent region 16B-1, but no fluorescence is generated in the second fluorescent region 16B-2. Therefore, the entire area of the observation object 15 is illuminated with a green striped surface light source.

  Here, when attention is paid to a region A2 facing the second fluorescent region 16B-2 in the object to be observed 15, the region A2 includes two first first regions located on both sides of the second fluorescent region 16B-2. The oblique illumination is performed by the fluorescence (green light) from the fluorescent region 16B-1.

  The fluorescence (green light) is high-intensity direct light (solid line) that passes through the area A2 as it is, and low-intensity scattered light that is scattered by the step in the refractive index (the edge of a phase object such as a cell) that exists in the area A2. The former is kicked by the imaging optical system 24 because most of the former have a large angle. Therefore, the image of the area A2 formed by the imaging optical system 24 becomes a dark field image, and the edge of the phase object existing in the area A2 appears in the dark field image.

  Note that the thickness (thickness in the optical axis direction) of the substrate body 16A is an optimum value that sufficiently increases the contrast of the dark field image (the relationship between the brightness of the background and the brightness of the edge image of the phase object). Is set in advance.

  On the other hand, when attention is paid to a region A1 facing the first fluorescent region 16B-1 in the observation object 15, the region A1 is front-facing by the fluorescence (green light) generated in the first fluorescent region 16B-1. Illuminated from.

  The fluorescence (green light) is also separated into high-intensity direct light (not shown) that passes through the area A1 as it is and low-intensity scattered light (not shown) scattered at the edge of the phase object existing in the area A1. However, most of the former cannot be kicked by the imaging optical system 24. Therefore, the image of the area A1 formed by the imaging optical system 24 is a bright field image, and the edge of the phase object existing in the area A1 does not appear in the bright field image.

  Note that the excitation light (blue light) that has passed through the first fluorescent region 16B-1 and the second fluorescent region 16B-2 as it is reaches the object 15 to be observed. Since it is cut before entering the imaging device 25 by 29-1, it can be ignored.

  Therefore, the state of the image of the observation object 15 at this time is as shown in FIG. FIG. 7 is a partial schematic diagram of an image of the observation object 15 when the wavelength of the excitation light (and the barrier light) is the same as the excitation wavelength (blue) of the first fluorescent region 16B-1. In FIG. 7, the area A1 'is an image of the area A1, and the area A2' is an image of the area A2. As shown in FIG. 7, since the area A2 'is a dark field image, the edge of the phase object appears as a bright green line on a dark background. However, since the area A1' is a bright field image, the area A1 'is almost uniformly bright green.

  FIG. 8 is a schematic diagram showing the state of the periphery of the observation object 15 when the wavelength of the excitation light (and the barrier light) is the same as the excitation wavelength (green) of the second fluorescent region 16B-2. In FIG. 8, symbol Lg indicates excitation light (green light), and symbol Lr indicates fluorescence (red light).

  As shown in FIG. 8, when the wavelength of the excitation light (and the barrier light) is the same as the excitation wavelength (green) of the second fluorescent region 16B-2, the second fluorescent region 16B-2 is excited and the first fluorescent region 16B. -1 is not excited. Therefore, fluorescence (red light) is generated in the second fluorescent region 16B-2, but no fluorescence is generated in the first fluorescent region 16B-1. Therefore, the entire area of the observation object 15 is illuminated with a red striped surface light source.

  Here, when attention is paid to a region A1 facing the first fluorescent region 16B-1 in the object 15 to be observed, the region A1 has two second regions located on both sides of the first fluorescent region 16B-1. Oblique illumination is performed by the fluorescence (red light) from the fluorescent region 16B-2.

  The fluorescence (red light) is separated into high-intensity direct light (solid line) that passes through the area A1 as it is, and low-intensity scattered light (dotted line) scattered at the edge of the phase object present in the area A1, Many of the former are kicked by the imaging optical system 24 because they have a large angle. Therefore, the image of the area A1 formed by the imaging optical system 24 becomes a dark field image, and the edge of the phase object existing in the area A1 appears in the dark field image.

  On the other hand, when attention is paid to a region A2 facing the second fluorescent region 16B-2 in the observation object 15, the region A2 is front-faced by the fluorescence (red light) generated in the second fluorescent region 16B-2. Illuminated from.

  The fluorescence (red light) is also separated into high-intensity direct light (not shown) that passes through the region A2 as it is and low-intensity scattered light (not shown) scattered at the edge of the phase object existing in the region A2. However, most of the former cannot be kicked by the imaging optical system 24. Therefore, the image of the region A2 formed by the imaging optical system 24 is a bright field image, and the edge of the phase object existing in the region A2 does not appear in the bright field image.

  Note that the excitation light (green light) that has passed through the first fluorescent region 16B-1 and the second fluorescent region 16B-2 as it is reaches the object 15 to be observed. Since it is cut by 29-2 before entering the image sensor 25, it can be ignored.

  Therefore, the state of the image of the observation object 15 at this time is as shown in FIG. FIG. 9 is a partial schematic diagram of an image of the observation object 15 when the wavelength of the excitation light (and the barrier light) is the same as the excitation wavelength (green) of the second fluorescent region 16B-2. In FIG. 9, a region A1 'is an image of the region A1, and a region A2' is an image of the region A2. As shown in FIG. 9, since the region A1 'is a dark field image, the edge of the phase object appears as a bright red line on a dark background, but the region A2' is a bright field image, so it is almost uniformly bright red.

  Therefore, if the image of the observation object 15 in the state shown in FIG. 7 and the image of the observation object 15 in the state shown in FIG. 9 are combined, all the phase objects included in the observation object 15 are visualized. be able to.

  FIG. 10 is an operation flowchart of the observation apparatus of the present embodiment. Steps S10, S15, and S16 in FIG. 10 are processes by the computer 37, and steps S11 to S14 are processes by the controller 36. Here, the operation of the computer 37 and the operation of the controller 36 are not distinguished from each other. Each step will be described as an operation.

  Step S10: The observation device monitors whether or not an observation start instruction is input from the user based on the signal from the input device 55. If a start instruction is input, the process proceeds to step S11. If not input, the process waits as it is.

  Step S11: The observation apparatus sets the combination of the excitation filter and the barrier filter inserted in the optical path to the combination of the first excitation filter 28-1 and the first barrier filter 29-1.

Step S12: observation apparatus is configured to light the light source 26 for a predetermined time period in this state, obtains the image data I ₁ of the one frame by driving the imaging element 25 within that period. The image data I ₁ is a green monochrome image (see FIG. 7).

  Step S13: The observation apparatus switches the combination of the excitation filter and the barrier filter inserted into the optical path to the combination of the second excitation filter 28-2 and the second barrier filter 29-2.

Step S14: observation apparatus is configured to light the light source 26 in this state for a predetermined period, and acquires the image data I ₂ of one frame by driving the imaging element 25 within that period. The image data I ₂ is a red monochrome image (see FIG. 9).

Step S15: observation apparatus performs color conversion processing on at least one of the image data I ₁ and the image data I _2. Characteristics of the color conversion processing, the hue of the image data I ₁ (green), the hue of a characteristic as to match the image data I ₂ and hues (red), for example, areas in the image data I ₁ A1 ' the average value is set based on the relationship between the hue average value of the region A2 'in the image data I _2. This relationship is determined by the relationship between the fluorescence wavelength of the first fluorescence region 16B-1 and the fluorescence wavelength of the second fluorescence region 16B-2, and the relationship basically does not change with time. Therefore, the characteristic of this color conversion process may be a fixed value.

Also, the observation apparatus performs a gradation conversion processing on at least one of the image data I ₁ and the image data I _2. The characteristics of the gradation conversion process are characteristics that match the gradation range of the image data I _{1 and} the gradation range of the image data I _2. For example, the gradation range of the image data I ₁ (maximum luminance) and minimum luminance) to be set based on the relationship between the gradation range of the image data I ₂ (maximum luminance and minimum luminance). This relationship is determined by the relationship between the fluorescence intensity of the first fluorescence region 16B-1 and the fluorescence intensity of the second fluorescence region 16B-2, and the relationship may change over time. Therefore, it is desirable that the characteristics of the gradation conversion process be updated periodically (or as necessary).

As a result of the color conversion processing and gradation conversion processing, the hue and tonal range of the image data I ₁ are consistent with the hue and tonal range of the image data I _2.

Step S16: observation device, with cut out 'image data I ₁ of the formation region of the dark-field image from the image data I ₁ (i.e. regions _A2)', formed of night vision field image from the image data I ₂ area (i.e. area A1 The image data I ₂ 'of') is cut out. Furthermore, the observation device, by joining image data I ₁ cut out from the image data I _{1 'and} the image data I ₂ cut out from the image data I _2' and generates image data I for one sheet, displays it Is converted into image data for use and then sent to the monitor 54. Therefore, the user can perform dark field observation of the entire area of the observation object 15 on the monitor 54 at a time. Note that the observation apparatus can also store the image data I generated in this step in accordance with an instruction from the user.

  As described above, in the observation apparatus of the present embodiment, the fluorescent member (the transparent fluorescent substrate 16 having the fluorescent layer 16B) is disposed near the object 15 and the fluorescent member is illuminated from a remote position. As a result, the object to be observed 15 is indirectly subjected to stripe illumination. Therefore, the observation apparatus of the present embodiment does not require a large apparatus around the object to be observed 15. In addition, since the light source 26 that can be a heat source can be separated from the object 15 to be observed, the state of the object 15 to be observed can be kept good.

  Moreover, since the fluorescent member (transparent fluorescent substrate 16) of the observation apparatus according to the present embodiment alternately arranges the first fluorescent regions 16B-1 and the second fluorescent regions 16B-2, excitation light (and barrier light). It is possible to change the phase of the stripe illumination simply by switching the wavelength.

  Further, since the fluorescent member (transparent fluorescent substrate 16) of the observation apparatus of the present embodiment is configured separately from the mechanism part of the observation apparatus (the main body of the stage 22), the fluorescent member has changed over time (discolored). In that case, it is only necessary to replace the fluorescent member with a new one, and there is no need to replace the mechanism (the main body of the stage 22) with a new one.

[Modification of First Embodiment]
Note that the observation object 15 of the observation apparatus of the present embodiment is a dish containing a culture solution containing biological cells. However, other types of observation objects, such as preparations dropped with a culture solution containing biological cells, A flask containing a culture solution containing living cells may also be used. However, since the thickness of the bottom of the object to be observed 15 may vary depending on the type of the object to be observed 15, in the observation apparatus of this embodiment, the distance between the fluorescent member (transparent fluorescent substrate 16) and the object to be observed 15. It is desirable that adjustment is possible.

  Incidentally, as a method for adjusting the distance between the fluorescent member (transparent fluorescent substrate 16) and the object 15 to be observed, for example, the substrate body 16A of the fluorescent member (transparent fluorescent substrate 16) is set thin in advance, and the fluorescence There is a method in which the user inserts one or a plurality of correction plates between the member (transparent fluorescent substrate 16) and the observation object 15 as necessary. This correction plate is a parallel flat plate (such as a glass substrate) that is transparent to visible light, similarly to the substrate body 16A.

  In the observation apparatus of the present embodiment, the two excitation wavelengths of the fluorescent member (transparent fluorescent substrate 16) are a combination of blue and green, but other combinations of colors (for example, a combination of purple and green) are used. There may be.

  In the observation apparatus of the present embodiment, the two types of excitation wavelengths of the fluorescent member (transparent fluorescent substrate 16) are both visible light, but at least one of the excitation wavelengths is a wavelength in a non-visible light region (for example, ultraviolet light). It is good.

  Further, in the observation apparatus of the present embodiment, the mounting destination of the fluorescent member (transparent fluorescent substrate 16) is the same as the mounting destination of the observation object 15 (stage 22), but may be separate. For example, as shown in FIG. 11, the mounting destination of the fluorescent member (transparent fluorescent substrate 16) may be the stage 22, and the observation object 15 may be supported by the transfer arm 30. In this case, the distance between the fluorescent layer 16B and the observation object 15 can be adjusted only by relatively moving the transfer arm 30 and the stage 22 in the optical axis direction.

  In the observation apparatus of the present embodiment, the position of the fluorescent member (the transparent fluorescent substrate 16 having the fluorescent layer 16B) in the optical axis direction with respect to the imaging optical system 24 is variable, but may be invariable. However, it is desirable that the positional relationship of the observation object 15 with respect to the imaging optical system 24 in the optical axis direction is variable.

In addition, in the observation device of the present embodiment, the color conversion process for matching the hue of the image data I _{1 and} the hue of the image data I ₂ is performed in step S15 described above. If it is allowed to appear, the color conversion process may be omitted.

In addition, in the observation device of the present embodiment, the gradation conversion process for matching the gradation range of the image data I _{1 and} the gradation range of the image data I ₂ is performed in step S15 described above. If it is allowed that a gradation step appears above, the gradation conversion process may be omitted.

  In the observation apparatus of the present embodiment, a color image sensor is used as the image sensor 25, but a monochrome image sensor may be used. In that case, even if the color conversion process is not performed in step S15, a step of hue does not appear on the final image data.

In the observation apparatus according to the present embodiment, the excitation light transmitted through the transparent fluorescent substrate 16 is prevented from entering the image sensor 25 by the barrier filter. However, the excitation light may be allowed to enter the image sensor 25. . However, in that case, it is necessary to use a color image sensor as the image sensor 25 in order to distinguish excitation light and fluorescence. In this case, since the entire image data I ₁ is blue and the entire image data I ₂ is green, color correction processing for removing the coloring may be added to the image processing in step S15 described above. desirable.

  Moreover, although the observation apparatus of this embodiment irradiated the excitation light to the transparent fluorescent substrate 16 from the front, you may irradiate from the diagonal direction. In that case, the excitation light transmitted through the transparent fluorescent substrate 16 can be prevented from entering the imaging optical system 24 without a barrier filter.

  In addition, two types of band-like fluorescent regions (first fluorescent region 16B-1 and second fluorescent region 16B-2) having different excitation wavelengths are alternately arranged on the fluorescent member (transparent fluorescent substrate 16) of the observation apparatus of the present embodiment. However, one fluorescent region may be omitted. However, in that case, in order to change the phase of the stripe illumination, it is necessary to change the positional relationship between the observation object 15 and the fluorescent member in the width direction of the fluorescent region. Therefore, in this case, a mechanism for separating the mounting destination of the fluorescent member and the mounting destination of the observation object 15 and shifting at least one of the fluorescent member and the observation object 15 in the direction perpendicular to the optical axis is necessary. It is.

Moreover, the observation apparatus of this embodiment may be combined with a culture apparatus. The culture apparatus is an apparatus that stores therein an observation container containing a culture solution containing living cells, and maintains the temperature, CO ₂ concentration, and the like of the observation container at predetermined values. If the observation container accommodated in the culture apparatus is transported to the observation apparatus of the present embodiment by the transport arm, the entire state of the observation container can be observed. Note that the observation apparatus may be incorporated inside the culture apparatus or disposed outside the culture apparatus. Incidentally, if the observation apparatus is incorporated in the culture apparatus, it is possible to perform observation without exposing the observation container to the outside air.

[Second Embodiment]
Hereinafter, embodiments of the observation container will be described.

  FIG. 12 is a schematic cross-sectional view of the observation container of the present embodiment. As shown in FIG. 12, the observation container of the present embodiment is a dish, and includes a container body 45A having a disk-shaped bottom part and a cylindrical side wall part, and a lid that covers a circular opening facing the bottom part of the container body 45A. Part 45B. Note that both the container main body 45A and the lid 45B are made of a material (resin or the like) that is transparent to visible light.

  A fluorescent layer 45C is provided on the surface of the bottom of the container main body 45A. In the fluorescent layer 45C, the same first fluorescent regions and second fluorescent regions as the fluorescent layer 16B of the first embodiment are alternately arranged without gaps. And the thickness of the bottom part of the container main body 45A is preset to the same optimum value as the thickness of the substrate main body 16A in the first embodiment.

  When such a dish is used together with the observation apparatus of the first embodiment, the transparent fluorescent substrate 16 loaded in the hollow portion of the stage 22 does not need to be provided with the fluorescent layer 16B, and the thickness thereof is also arbitrary. It doesn't matter. In this case, the bottom of the dish plays the same role as the transparent fluorescent substrate 16 of the first embodiment.

  Although the dish has been described here as an observation container, it may be another culture container such as a flask as long as it has a uniform thickness and a flat part (part of a parallel plate). . In any case, the formation destination of the fluorescent layer 45C is the surface of a flat portion having a uniform thickness. In either case, the observation is performed with the surface on which the fluorescent layer 45C is formed facing the illumination optical system 27 described above.

  Further, the fluorescent layer 45C of the present embodiment can be modified in the same manner as the fluorescent layer 16B of the first embodiment.

[Third Embodiment]
Hereinafter, embodiments of the preparation will be described.

  FIG. 13 is a schematic cross-sectional view of the preparation of the present embodiment. As shown in FIG. 13, the preparation of the present embodiment includes a slide glass 50 and a cover glass 51, and both the slide glass 50 and the cover glass 51 are made of a material (such as glass) that is transparent to visible light.

  A fluorescent layer 50B is provided on one surface of the slide glass 50, and a culture solution 61 containing biological cells is dropped on the other surface of the slide glass 50, and the cover glass 51 is in close contact therewith. Yes. Among them, the fluorescent layer 50B has the same first fluorescent regions and second fluorescent regions as the fluorescent layer 16B of the first embodiment, which are alternately arranged without gaps, and the thickness of the slide glass 50 is the same as that of the first embodiment. Is set to the same optimum value as the thickness of the substrate body 16A.

  When such a preparation is used together with the observation apparatus of the first embodiment, the transparent fluorescent substrate 16 loaded in the hollow portion of the stage 22 does not need to be provided with the fluorescent layer 16B, and the thickness thereof is also arbitrary. I do not care. In this case, the slide glass 50 plays the same role as the transparent fluorescent substrate 16 of the first embodiment.

  In addition, although the formation destination of the fluorescent layer 50B in this embodiment was the slide glass 50 side, it may be the cover glass 51 side. However, in that case, the thickness of the slide glass 50 is arbitrary, and the thickness of the cover glass 51 is set to the same optimum value as the thickness of the substrate body 16A in the first embodiment. In either case, the observation is performed with the surface on which the fluorescent layer 50B is formed facing the illumination optical system 27 side.

  Also, the fluorescent layer 50B of the present embodiment can be modified in the same manner as the fluorescent layer 16B of the first embodiment.

[Supplement of means for solving problems]
In the aspect of the observation apparatus described above, second fluorescence that emits fluorescence in the predetermined wavelength range in the gap between the first fluorescent portions of the fluorescent member according to light having a second wavelength different from the first wavelength. And a switching unit that switches the wavelength of light that illuminates the fluorescent member between the first wavelength and the second wavelength.

  The predetermined wavelength range may be a visible light range.

  Further, in the above-described one aspect of the observation container, in the gap between the first fluorescent portions in the bottom portion, the second fluorescence that emits fluorescence in the predetermined wavelength range according to light having a second wavelength different from the first wavelength. A fluorescent part may be provided.

  The predetermined wavelength range may be a visible light range.

  Further, in one aspect of the slide glass described above, a second fluorescent part that emits fluorescence within the predetermined wavelength range is provided in the gap between the first fluorescent parts in response to light having a second wavelength different from the first wavelength. May be.

  The predetermined wavelength range may be a visible light range.

It is a block diagram of the optical system part of the observation apparatus of 1st Embodiment. It is a block diagram of the circuit part of the observation apparatus of 1st Embodiment. It is a figure explaining the transparent fluorescent substrate 16 of 1st Embodiment in detail. It is a figure which shows the relationship between the characteristic of 1st fluorescence area | region 16B-1, and the characteristic of the 1st excitation filter 28-1. It is a figure which shows the relationship between the characteristic of 2nd fluorescence area | region 16B-2, and the characteristic of the 2nd excitation filter 29-2. It is a schematic diagram which shows the mode of the periphery of the to-be-observed object 15 when the wavelength of excitation light (and barrier light) is the same as the excitation wavelength (blue) of 1st fluorescence area | region 16B-1. It is a partial schematic diagram of the image of the observation object 15 when the wavelength of the excitation light (and the barrier light) is the same as the excitation wavelength (blue) of the first fluorescent region 16B-1. It is a schematic diagram which shows the mode of the periphery of the to-be-observed object 15 when the wavelength of excitation light (and barrier light) is the same as the excitation wavelength (green) of 2nd fluorescence area | region 16B-2. It is a partial schematic diagram of the image of the object to be observed 15 when the wavelength of the excitation light (and barrier light) is the same as the excitation wavelength (green) of the second fluorescent region 16B-2. It is an operation | movement flowchart of the observation apparatus of 1st Embodiment. It is a figure explaining the modification of 1st Embodiment. It is a schematic sectional drawing of the observation container of 2nd Embodiment. It is a schematic sectional drawing of the preparation of 3rd Embodiment.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 26 ... Light source, 27 ... Illumination optical system, 28 ... Turret, 22 ... Stage 22, 24 ... Imaging optical system, 29 ... Turret, 25 ... Imaging element, 16 ... Transparent fluorescent substrate 16B ... Fluorescent layer

Claims (9)

An observation apparatus for observing an object to be observed transparent to light in a predetermined wavelength range,
A fluorescent member in which a first fluorescent part that emits fluorescence within the predetermined wavelength range in response to light of a first wavelength is repeatedly provided;
An illumination optical system for illuminating the fluorescent member with light of the first wavelength;
An observation optical system for generating an image of the observation object illuminated with fluorescence emitted by the fluorescent member;
An observation apparatus comprising: The observation apparatus according to claim 1,
In the gap of the first fluorescent part of the fluorescent member,
A second fluorescent part that emits fluorescence within the predetermined wavelength range in response to light having a second wavelength different from the first wavelength;
An observation apparatus, further comprising switching means for switching a wavelength of light that the illumination optical system illuminates the fluorescent member between the first wavelength and the second wavelength. The observation apparatus according to claim 2,
The observation device, wherein the predetermined wavelength range is a visible light range. An observation container that is transparent to light in a predetermined wavelength range,
At the bottom of the observation container,
An observation container, wherein a first fluorescent part that emits fluorescence within the predetermined wavelength range according to light of a first wavelength is repeatedly provided at intervals. The observation container according to claim 4,
In the gap of the first fluorescent part in the bottom,
An observation container, comprising: a second fluorescent part that emits fluorescence in the predetermined wavelength range in response to light having a second wavelength different from the first wavelength. The observation container according to claim 5,
The said predetermined wavelength range is a visible light range. The observation container characterized by the above-mentioned. A slide glass that is transparent to wavelengths within a predetermined wavelength range,
A slide glass, wherein a first fluorescent part that emits fluorescence in the predetermined wavelength range according to light of a first wavelength is repeatedly provided at intervals. The slide glass according to claim 7,
In the gap between the first fluorescent parts,
A slide glass comprising a second fluorescent part that emits fluorescence in the predetermined wavelength range in response to light having a second wavelength different from the first wavelength. The slide glass according to claim 8,
The slide glass characterized in that the predetermined wavelength range is a visible light range.

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