JP2006011045A - Total reflection microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a total reflection microscope in which the state of the inside of a stage cover can be visually observed. <P>SOLUTION: The total reflection microscope is equipped with an inverted microscope, a total reflection illumination optical system for total reflection illumination, the stage cover constituted of a fixed cover 124 and an attachable/detachable cover 125, and an auxiliary illumination part. The inverted microscope is equipped with a stage for placing a dish 113 in which a sample 112 is put, an objective lens 110 positioned under the dish 113, and an observation optical system for observing the sample 112. The observation optical system is equipped with the objective lens 110, an absorption filter 121, an image-formation lens 122 and a CCD camera 123. The attachable/detachable cover 125 is attached to/detached from the fixed cover 124, and a band-pass filter 126 is fixed at the upper opening part of the attachable/detachable cover 125. The auxiliary illumination part has an LED head 132. The transmission area of the absorption filter 121 in the observation optical system is not overlapped on the transmission area of the band-pass filter 126. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a total reflection microscope for performing fluorescence observation using evanescent light generated by total reflection illumination.

  In recent years, a microscope method called total internal reflection fluorescence microscopy (TIRFM) has attracted attention as a fluorescence observation method of a biological microscope. This is a method of exciting a fluorescent substance using light called evanescent light that immerses in a slight range of about several hundreds of nanometers on the specimen side when the illumination light is totally reflected at the interface between the cover glass and the specimen. In addition, since only a small range of fluorescent material in the vicinity of the cover glass is illuminated, the background is very dark, and high-contrast fluorescent observation and weak fluorescent observation are possible.

  Generally, a laser light source that emits monochromatic light with sufficient intensity is used as a light source for a total reflection fluorescent microscope. In an actual apparatus, there is a possibility that direct refracted light or scattered light leaks to the outside due to the change of the revolver in normal operation or other misoperations, resulting in no total reflection at the interface between the cover glass and the specimen.

  For this reason, in consideration of user safety, the user can use the stage cover to cover the entire stage so that direct refracted light and scattered light do not leak to the outside in every state that can occur during user operations. Is provided. When changing the specimen or spotting oil, if the lid of the stage cover is opened, the shutter enters the laser beam path and the introduction of laser light to the specimen is blocked. On the other hand, when the lid of the stage cover is closed, the shutter is opened in the laser beam path, and the laser beam is introduced into the specimen.

  Japanese Patent Application Laid-Open No. 11-326774 discloses a microscope light source centering device. In this apparatus, a light source image on the screen surface can be observed by attaching a cylindrical body made of transparent plastic having a screen on the bottom surface to the revolver.

Japanese Laid-Open Patent Publication No. 10-83060 discloses a clear vision device for a dark box. In this apparatus, an infrared illuminator and a CCD camera are provided inside a dark box, which is a work space, and the inside of the dark box can be confirmed by a monitor disposed outside.
JP-A-11-326774 Japanese Patent Laid-Open No. 10-83060

  However, the prior art has the following problems.

  During the total reflection fluorescence observation in which laser light is introduced into the specimen, the stage is covered with a stage cover, so the state inside the stage cover is unknown. In this state, if the positional relationship between the objective lens and the specimen is greatly changed by operating the stage, the specimen or stage center may come into contact with the tip of the objective lens or condenser lens.

  In the apparatus disclosed in Japanese Patent Laid-Open No. 11-326774, only the cylindrical body mounted on the revolver is used, and the entire upper surface of the stage is not covered.

  In the apparatus disclosed in Japanese Patent Laid-Open No. 10-83060, a dedicated CCD camera inside the dark box and a dedicated monitor outside are necessary, and the system becomes expensive.

  The present invention has been made in consideration of such a situation, and an object of the present invention is to provide a total reflection microscope capable of visually observing the inside of the stage cover during total reflection fluorescence observation. .

  The total reflection microscope of the present invention includes a microscope having a stage for placing a sample, a total reflection illumination optical system for total reflection illumination of the sample, a stage cover provided to cover the stage of the microscope, And an auxiliary illumination light source unit that illuminates the interior of the stage cover with light of a monochromatic wavelength. The microscope has an observation optical system for observing the specimen, and the observation optical system includes an objective lens and an absorption filter. The stage cover includes a fixed cover and a detachable cover. At least a part of the auxiliary illumination light source unit is fixed to the fixed cover. The detachable cover has a bandpass filter that allows an observer to visually observe the inside of the stage cover. The bandpass filter transmits monochromatic wavelength light emitted from the auxiliary illumination light source unit, but is totally reflected. The light of the optical system is not transmitted, and the transmission range of the bandpass filter does not overlap with the transmission range of the absorption filter in the observation optical system.

  ADVANTAGE OF THE INVENTION According to this invention, the total reflection microscope which can visually observe the state inside a stage cover at the time of total reflection fluorescence observation is provided.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[First embodiment]
FIG. 1 is a longitudinal sectional view of an inverted total reflection fluorescent microscope according to the first embodiment of the present invention.

  The inverted total reflection fluorescent microscope of this embodiment includes an inverted microscope, a total reflection illumination optical system for total reflection illumination of a specimen, an interlock unit, and an auxiliary illumination unit.

  The inverted microscope is a stage for placing a dish 113 containing a specimen 112, an objective lens 110 positioned below the dish 113 placed on the stage, and a specimen 112 including the objective lens 110. An observation optical system, a revolver 111 for holding the objective lens 110, and a semi-focus mechanism (not shown) for moving the revolver 111 along the optical axis T of the objective lens 110 are provided.

  The objective lens 110 is attached to the revolver 111. The revolver 111 is movable along the optical axis T of the objective lens 110 by a semi-focusing mechanism.

  The stage includes an upper stage 117, a lower stage 118, and a stage fixing unit 119. The upper stage 117 is connected to the lower stage 118 via a ball guide mechanism (not shown) or the like. The lower stage 118 is connected to the stage fixing part 119 via a ball guide mechanism (not shown) or the like. The lower stage 118 is movable in a direction perpendicular to the paper surface within a plane perpendicular to the optical axis T of the objective lens. The upper stage 117 is movable in the horizontal direction on the paper surface within a plane perpendicular to the optical axis T of the objective lens.

  A center seat 116 is provided at the opening in the center of the upper stage 117. Therefore, the center seat 116 can move in a plane perpendicular to the optical axis T of the objective lens 110. A dish 113 containing a specimen 112 is placed on the center seat 116. The dish 113 is made of transparent plastic, and has a cover glass 114 that blocks an opening formed by hollowing out the bottom surface. The specimen 112 is cultured on the upper surface of the cover glass 114. The inside of the dish 113 is immersed in the culture solution 115. Oil 120 is immersed between the tip of the objective lens 110 and the cover glass 114.

  The observation optical system includes an objective lens 110, an absorption filter 121, an imaging lens 122, and a CCD camera 123. The absorption filter 121, the imaging lens 122, and the CCD camera 123 are arranged in order from the objective lens 110 on the optical axis T of the objective lens 110.

  The total reflection illumination optical system includes a laser head 101, a laser projection tube 107, and an objective lens 110 in the observation optical system. The interlock section includes a stage cover including a fixed cover 124 and a detachable cover 125 and an interlock box 102.

  An interlock box 102 is fixed to the laser head 101 with screws (not shown) or the like, and a fiber fixing portion 103 is fixed to the interlock box 102 with screws (not shown). The fiber fixing portion 103 is provided with an introduction portion 104 of a fiber 105, and the emission portion 106 at the other end of the fiber 105 is fixed to the laser projection tube 107 with screws (not shown). The laser projection tube 107 includes a condensing lens 108 and a reflection mirror 109 inside. The condensing lens 108 has a focal length that condenses the light emitted from the fiber 105 at the rear focal position of the objective lens 110. The optical axis M of the laser projection tube 107 is orthogonal to the optical axis T of the objective lens 110. The reflection mirror 109 has an angle of 45 degrees with respect to the optical axis M of the laser projection tube 107, and reflects the light introduced into the laser projection tube 107 toward the outer periphery of the objective lens 110. Has been placed. The reflection mirror 109 can be moved around the outer periphery of the objective lens 110 in the direction of the optical axis M of the laser projection tube 107 by a linear motion mechanism (not shown) and a spring (not shown).

  A fixed cover 124 made of metal such as aluminum is fixed to the upper stage 117 with screws (not shown) or the like so as to cover almost the entire upper surface of the upper stage 117. The fixed cover 124 has an opening at the top. On the fixed cover 124, a metal detachable cover 125 such as aluminum is attached. The detachable cover 125 can be attached to and detached from the fixed cover 124. The detachable cover 125 has an opening at the top, and a band pass filter 126 is fixed to the opening by bonding or the like. When the detachable cover 125 is attached to the fixed cover 124, the detachable cover 125 and the fixed cover 124 are integrated into a box shape. A switch 127 is fixed to the inner surface of the fixed cover 124 with a screw (not shown) or the like. The switch 127 has a lever 128 for ON / OFF switching. Pins 129 are fixed to the back surface of the detachable cover 125 with screws (not shown). The pin 129 pushes the lever 128 of the switch 127 when the detachable cover 125 is attached to the fixed cover 124. A cable 130 for transmitting an ON / OFF signal is connected to the switch 127. An ON / OFF signal from the switch 127 is sent to a circuit (not shown) inside the interlock box 102 through the cable 130 to open and close a shutter 131 driven by an internal motor (not shown). .

  The auxiliary illumination unit includes an LED head 132 that is a small LED light source and a hand switch 134 that is connected to the LED head 132 via a cable 133. The LED head 132 is provided on the inner surface on the rear side of the fixed cover 124. The illumination light emitted from the LED head 132 is monochromatic wavelength light and illuminates the inside of the stage cover (fixed cover 124 and detachable cover 125). Specifically, the dish 113, the center seat 116, and the tip of the objective lens 110 are illuminated. The LED head 132 can be turned on / off by a hand switch 134 connected via a cable 133.

  The wavelength of monochromatic light from the laser head 101 in the total reflection illumination optical system is λL, the wavelength of monochromatic light from the LED head 132 is λ0, the transmission region of the absorption filter 121 is λ1 to λ2, and the transmission region of the bandpass filter 126 is Let λ3 to λ4. The transmission regions λ1 to λ2 of the absorption filter 121 in the observation optical system and the transmission regions λ3 to λ4 of the bandpass filter 126 do not overlap. The band pass filter 126 transmits monochromatic light from the LED head 132. That is, λ3 <λ0 <λ4. Further, the band pass filter 126 does not transmit monochromatic light from the laser head 101. That is, λL <λ3 or λ4 <λL.

  Next, the operation of this embodiment will be described.

  When the detachable cover 125 is attached to the fixed cover 124, the pin 129 pushes the lever 128 of the switch 127 and an ON signal is output from the switch 127. The ON signal is sent to a circuit inside the interlock box 102 through the cable 130 to open the shutter 131. The laser beam from the laser head 101 is introduced into the fiber 105, collected by the condenser lens 108 of the laser projection tube 107, reflected by the reflection mirror 109, and then illuminates the specimen 112 in the total reflection state. The evanescent light is generated near the boundary surface by the total reflection illumination, and the generated evanescent light excites the specimen 112. The specimen 112 excited by the evanescent light emits fluorescence. The fluorescence from the specimen 112 is collected by the objective lens 110, unnecessary light is cut by the absorption filter 121, and is imaged on the light receiving surface of the CCD 123 by the imaging lens 122.

  In such a total reflection fluorescent illumination observation state, the hand switch 134 is turned on to illuminate with the LED head 132. Thus, the observer 135 can visually observe the inside of the stage cover (the fixed cover 124 and the detachable cover 125) through the bandpass filter 126. Specifically, the observer 135 can grasp the positional relationship among the objective lens 110, the dish 113, and the center seat 116. Therefore, when the center seat 116 or the semi-focus portion is moved, the positional relationship between the outer peripheral portion of the tip of the objective lens 110 and the inner wall surface of the central opening of the center seat 116, the objective lens 110 lifts the bottom surface of the dish 113. Can recognize whether or not.

  As described above, in the present embodiment, in the inverted total reflection fluorescent microscope, the periphery of the tip of the objective lens can be visually observed, so the tip of the objective lens and the bottom surface of the dish, the side surface of the tip of the objective lens, and the middle Contact with the inner wall surface of the seat can be avoided. In addition, the stage can be moved and focused quickly, leading to improved workability.

[Second Embodiment]
FIG. 2 is a longitudinal sectional view of an upright total reflection fluorescence microscope according to the second embodiment of the present invention. In FIG. 2, members indicated by the same reference numerals as those shown in FIG. 1 are similar members, and detailed description thereof is omitted.

  The upright total reflection fluorescent microscope of this embodiment includes a stage-fixed upright microscope, a total reflection illumination optical system for total reflection illumination of a specimen, an interlock unit, and an auxiliary illumination unit. .

  The erecting microscope is for observing the stage 112 on which the dish 113 containing the specimen 112 is placed, the objective lens 110 located above the dish 113 placed on the stage, and the specimen 112 including the objective lens 110. And an quasi-focus mechanism (not shown) for moving the objective lens 110 along the optical axis T.

  The configuration of the stage is the same as in the first embodiment. The configuration of the observation optical system is such that the absorption filter 121, the imaging lens 122, and the CCD camera 123 are arranged in this order from the objective lens 110 in the upward direction across the sample and the total reflection illumination optical system. ing.

  The total reflection illumination optical system includes a laser head 101, a laser projection tube 107, and a condenser lens 136. The interlock portion includes a stage cover including a fixed cover 137 and a detachable cover 138, and an interlock box 102.

  The configuration of the laser head 101 and the configuration of the interlock box 102 are the same as those in the first embodiment.

  The condenser lens 136 is positioned below the dish 113 placed on the stage and is fixed to the condenser base 141. The condenser base 141 is movable in the optical axis T direction of the condenser lens 136 by a rack and pinion method or the like, and is fixed to a stage fixed upright microscope. Oil 120 is immersed between the tip of the condenser lens 136 and the cover glass 114.

  The condensing lens 108 in the laser projection tube 107 has a focal length that condenses the light emitted from the fiber 105 at the rear focal position of the condenser lens 136. The optical axis M of the laser projection tube 107 is orthogonal to the optical axis T of the condenser lens 136. The reflection mirror 109 has an angle of 45 degrees with respect to the optical axis M of the laser projection tube 107 and reflects the light introduced into the laser projection tube 107 toward the outer peripheral portion of the condenser lens 136. Has been placed. The reflection mirror 109 can move around the outer periphery of the condenser lens 136 in the direction of the optical axis M of the laser projection tube 107 by a linear motion mechanism (not shown) and a spring (not shown).

  A fixed cover 137 made of metal such as aluminum is fixed to the upper stage 117 with screws (not shown) or the like so as to cover the rear surface of the upper stage 117. A metal detachable cover 138 such as aluminum is detachable from the fixed cover 137. The detachable cover 138 has an opening on the observer side, and a band pass filter 140 is fixed to the opening by bonding (not shown) or the like. When the detachable cover 138 is attached to the fixed cover 137, the detachable cover 138 and the fixed cover 137 are integrated to form a box shape that covers almost the entire upper surface of the upper stage 117 and the objective lens 110. A switch 127 is fixed to the fixed cover 137. The switch 127 has a lever 128 for ON / OFF switching. A pin 129 is fixed to the detachable cover 138 with a screw (not shown) or the like. The pin 129 pushes the lever 128 of the switch 127 when the detachable cover 138 is attached to the fixed cover 137. An ON / OFF signal from the switch 127 is sent to a circuit (not shown) inside the interlock box 102 through the cable 130 to open and close a shutter 131 driven by an internal motor (not shown). .

  The auxiliary illumination unit includes an LED head 132 that is a small LED light source and a hand switch 134 that is connected to the LED head 132 via a cable 133. The LED head 132 is provided on the inner surface on the rear side of the fixed cover 137. Illumination light emitted from the LED head 132 is monochromatic wavelength light and illuminates the inside of the stage cover (fixed cover 137 and detachable cover 138). Specifically, the dish 113, the center seat 116, and the tip of the objective lens 110 are illuminated. The LED head 132 can be turned on / off by a hand switch 134 connected via a cable 133.

  Next, the operation of this embodiment will be described.

  When the detachable cover 138 is attached to the fixed cover 137, the pin 129 pushes the lever 128 of the switch 127 and an ON signal is output from the switch 127. The ON signal is sent to a circuit inside the interlock box 102 through the cable 130 to open the shutter 131. The laser beam from the laser head 101 is introduced into the fiber 105, collected by the condenser lens 108 of the laser projection tube 107, reflected by the reflection mirror 109, and then illuminates the specimen 112 in the total reflection state. The evanescent light is generated near the boundary surface by the total reflection illumination, and the generated evanescent light excites the specimen 112. The specimen 112 excited by the evanescent light emits fluorescence. The fluorescence from the specimen 112 is collected by the objective lens 110, unnecessary light is cut by the absorption filter 121, and is imaged on the light receiving surface of the CCD 123 by the imaging lens 122.

  In such a total reflection fluorescent illumination observation state, the hand switch 134 is turned on to illuminate with the LED head 132. Thus, the observer 135 can visually observe the inside of the stage cover (the fixed cover 137 and the detachable cover 138) through the bandpass filter 140. Specifically, the observer 135 can grasp the positional relationship among the objective lens 110, the dish 113, and the center seat 116. Therefore, when the center seat 116 or the semi-focus portion is moved, the positional relationship between the outer peripheral portion of the tip of the objective lens 110 and the inner wall surface of the central opening of the center seat 116 and the positions of the objective lens 110 and the sample 112 are recognized. Can do.

  As described above, in the present embodiment, in the upright type total reflection fluorescent microscope, since the peripheral portion of the tip of the objective lens can be visually observed, the tip of the objective lens, the bottom surface of the dish, the side surface of the tip of the objective lens, Contact with the inner wall surface of the center seat can be avoided. In addition, the stage can be moved and focused quickly, leading to improved workability.

[Third embodiment]
FIG. 3 is a longitudinal sectional view of an inverted total reflection fluorescent microscope according to the third embodiment of the present invention. In FIG. 3, members indicated by the same reference numerals as those shown in FIG. 1 are similar members, and detailed description thereof is omitted.

  In the present embodiment, the configuration of the auxiliary illumination unit is different from that of the first embodiment.

  An LED head 132 is provided on the inner surface of the rear side of the fixed cover 124. The illumination light emitted from the LED head 132 illuminates the dish 113, the center seat 116, and the tip of the objective lens 110. The LED head 132 is connected to a circuit (not shown) of the interlock box 102 through a cable 142. The circuit inside the interlock box 102 sends an OFF signal to the LED head 132 through the cable 142 when the shutter 131 is closed, and sends an ON signal when the shutter 131 is open.

  Next, the operation of this embodiment will be described.

  The LED head 132 is turned ON / OFF in conjunction with the attachment / detachment of the attachment / detachment cover 125 to / from the fixed cover.

  As can be seen from the above, this embodiment has the advantage of saving the trouble of turning on / off the LED head 132 in addition to the advantages of the first embodiment. Thereby, total reflection fluorescence observation can be performed more rapidly.

  The present embodiment is an example applied to an inverted total reflection fluorescence microscope, but may be applied to the upright total reflection fluorescence microscope described in the second embodiment.

[Fourth embodiment]
FIG. 4 is a longitudinal sectional view of an inverted total reflection fluorescent microscope according to the fourth embodiment of the present invention. In FIG. 4, members indicated by the same reference numerals as those shown in FIG. 1 are similar members, and detailed description thereof is omitted.

  In the present embodiment, the configuration of the auxiliary illumination unit is different from that of the first embodiment.

  A bandpass filter 145 is provided on the rear side surface of the fixed cover 124. This band pass filter 145 has a transmission region similar to that of the band pass filter 126 of the removable cover 125. That is, the transmission region of the bandpass filter 145 is λ3 to λ4. Further, a halogen light source 144 that emits light of continuous wavelength is disposed with the beam emission side facing the band-pass filter 145 side. The halogen light source 144 is fixed to the outer side surface of the fixed cover 124 with a screw (not shown) or the like via the adapter 143.

  Next, the operation of this embodiment will be described.

  Light emitted from the halogen light source 144 passes through the band-pass filter 145 and becomes only light having a wavelength component of λ3 to λ4, and illuminates the dish 113, the center 116, and the tip of the objective lens 110. For this reason, the observer can grasp the positional relationship among the dish 113, the center seat 116, and the tip of the objective lens 110 through the band-pass filter 126.

  As described above, in this embodiment, the inside of the stage cover (fixed cover 124 and detachable cover 125) can be visually observed using a normal halogen light source that emits light of a continuous wavelength.

[Modification]
In the modification, another band pass filter is provided on the side surface of the fixed cover 124 on the viewer 135 side instead of the band pass filter 145. The bandpass filter has a transmission region similar to that of the bandpass filter 145. In addition, a dedicated auxiliary illumination light source such as a halogen light source 144 is not arranged, and the inside of the stage cover is illuminated through a band-pass filter with a flashlight or the like.

  In this modification, the internal state of the stage cover can be visually observed without the need for a dedicated auxiliary illumination light source.

  The present embodiment is an example applied to an inverted total reflection fluorescence microscope, but may be applied to the upright total reflection fluorescence microscope described in the second embodiment.

  The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. Also good.

1 is a longitudinal sectional view of an inverted total reflection fluorescence microscope according to a first embodiment of the present invention. It is a longitudinal cross-sectional view of an upright total reflection fluorescence microscope according to a second embodiment of the present invention. It is a longitudinal cross-sectional view of the inverted total reflection fluorescence microscope by 3rd embodiment of this invention. It is a longitudinal cross-sectional view of the inverted total reflection fluorescence microscope by 4th embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 ... Laser head, 102 ... Interlock box, 103 ... Fiber fixing part, 104 ... Introduction part, 105 ... Fiber, 106 ... Ejection part, 107 ... Laser projection tube, 108 ... Condensing lens, 109 ... Reflection mirror, 110 ... objective lens, 111 ... revolver, 112 ... specimen, 113 ... dish, 114 ... cover glass, 115 ... culture solution, 116 ... center, 117 ... upper stage, 118 ... lower stage, 119 ... stage fixing part, 120 ... oil 121 ... Absorption filter, 122 ... Imaging lens, 123 ... CCD camera, 124 ... Fixed cover, 125 ... Removable cover, 126 ... Band pass filter, 127 ... Switch, 128 ... Lever, 129 ... Pin, 130 ... Cable, 131 ... Shutter, 132 ... LED head, 133 ... Cable, 34 ... Hand switch, 135 ... Observer, 136 ... Condenser lens, 137 ... Fixed cover, 138 ... Removable cover, 140 ... Band pass filter, 141 ... Condenser base, 142 ... Cable, 143 ... Adapter, 144 ... Halogen light source, 145 ... bandpass filter.

Claims (6)

A microscope having a stage for placing a specimen;
A total reflection illumination optical system for total reflection illumination of the specimen;
A stage cover provided to cover the microscope stage;
An auxiliary illumination light source unit that illuminates the interior of the stage cover with light of a monochromatic wavelength,
The microscope has an observation optical system for observing the specimen, the observation optical system includes an objective lens and an absorption filter, the stage cover includes a fixed cover and a removable cover, and the auxiliary illumination light source unit is at least partly Is fixed to the fixed cover, and the detachable cover has a bandpass filter that allows an observer to visually observe the inside of the stage cover, and the bandpass filter has a monochromatic wavelength emitted by the auxiliary illumination light source unit. A total reflection microscope that transmits light but does not transmit light from the total reflection illumination optical system, and the transmission range of the bandpass filter does not overlap the transmission range of the absorption filter in the observation optical system.   2. The total reflection microscope according to claim 1, wherein the microscope is an inverted microscope, the inverted microscope has an objective lens positioned below a sample placed on a stage, and the total reflection illumination optical system includes an objective lens.   2. The total reflection according to claim 1, wherein the microscope is an upright microscope, the upright microscope has a condenser lens positioned below a specimen placed on the stage, and the total reflection illumination optical system includes a condenser lens. microscope.   The total reflection microscope according to claim 1, wherein the auxiliary illumination light source unit includes an LED light source, and the LED light source is fixed to a fixed cover.   5. The total reflection microscope according to claim 4, wherein the LED light source is turned on / off in conjunction with the attachment / detachment of the attachment / detachment cover to / from the fixed cover.   2. The auxiliary illumination light source unit according to claim 1, comprising a bandpass filter fixed to a fixed cover and a continuous wavelength light source that emits light of a continuous wavelength, and the bandpass filter of the fixed cover is a bandpass of the removable cover. A total reflection microscope that has a transmission region similar to that of a filter, and illuminates the inside of the stage cover with light of a continuous wavelength emitted from a continuous wavelength light source through a band-pass filter provided on the fixed cover.

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