JP5437052B2 - Microscope equipment - Google Patents

Description

  The present invention relates to a microscope apparatus.

  Conventionally, when the excitation light emitted from the light source is totally reflected at the boundary surface between the cover glass and the specimen, the fluorescent material is excited using evanescent light that oozes out to a slight range of about several hundred nm on the specimen side. A total reflection microscope for observing emitted fluorescence is known (for example, see Patent Document 1). In the total reflection microscope, excitation light that has become substantially parallel light is incident on the specimen from the off-axis. In Patent Document 1, when the excitation light is not totally reflected at the interface between the cover glass and the specimen due to an erroneous operation of the specimen or the revolver, the direct refracted light or scattered light is emitted in an unexpected direction. In order to prevent this, the entire specimen is covered with a cover. Fluorescence can be observed through a bandpass filter provided on the cover while preventing the excitation light that is totally reflected by the cover from being emitted to the outside.

JP 2006-11045 A

However, when the cover is placed on the specimen, there is a disadvantage that the transmitted light in the specimen of the excitation light cannot be observed.
That is, by switching the incident light beam to the objective lens, it is possible to easily switch between transmitted light observation and total reflection illumination observation, but when switching to transmitted light observation, it is necessary to remove the cover from the specimen. When switching to reflected illumination observation, it is necessary to cover the specimen with the cover.

  For this reason, when switching from total reflection illumination observation to transmitted light observation, the observer must remove the cover after confirming that the excitation light for total reflection illumination has been blocked. When removed, there is a disadvantage that the excitation light is emitted in an unexpected direction. Even when switching from transmitted light observation to total reflection illumination observation, it is necessary to confirm that the excitation light for total reflection illumination is blocked until the cover is put on. Therefore, the observer has to perform troublesome confirmation work, and there is an inconvenience that ease of handling and functionality are impaired.

  The present invention has been made in view of the above-described circumstances, and provides a microscope apparatus capable of easily switching between total reflection illumination observation and transmitted light observation without performing a troublesome confirmation operation by an observer. It is aimed.

In order to achieve the above object, the present invention provides the following means.
According to the present invention, an objective lens disposed opposite to a specimen, and illumination light composed of substantially parallel light is incident on an entrance pupil position of the objective lens, and the illumination light is incident on the specimen by the objective lens. A scanning illumination optical system that condenses and scans, a total reflection illumination optical system that condenses illumination light off the axis of the entrance pupil position of the objective lens, and makes evanescent light incident on the specimen, and the total reflection illumination optics An incident switching means for switching incidence of the illumination light to the objective lens via the system or the scanning illumination optical system, and a transmission detection optical system for detecting illumination light scanned by the scanning illumination optical system and transmitted through the specimen, An openable / closable cover member disposed on the opposite side of the objective lens across the specimen, an open / close sensor for detecting the open / close of the cover member, and the open / close sensor opens the cover member. When issued, to provide a microscope apparatus and a shutter for blocking the incident to the objective lens of the illumination light via total internal reflection illumination optical system.

  According to the present invention, when the optical system through which the illumination light is incident is switched to the total reflection illumination optical system by the incident switching means, the opening / closing sensor detects the opening / closing of the cover member and the cover is detected to be in the open state. The shutter blocks the illumination light from entering the objective lens via the total reflection illumination optical system. When the closed state of the cover member is detected, the illumination light is allowed to enter the objective lens via the total reflection illumination optical system, and the illumination light is collected off-axis at the entrance pupil position of the objective lens. As a result, total reflection occurs in the specimen, and evanescent light is incident. Thereby, total reflection illumination observation can be performed.

On the other hand, when the optical system for making the illumination light incident is switched to the scanning illumination optical system by the incident switching means, the illumination light incident on the objective lens via the scanning illumination optical system is condensed and scanned on the specimen, and the specimen is scanned. When the transmitted illumination light is detected by the transmission detection optical system, it is possible to perform transmission observation of the specimen.
In this case, according to the present invention, when the cover member is opened, the illumination light is not automatically incident on the objective lens via the total reflection illumination optical system by the shutter. It is possible to prevent inconvenience that the light is emitted in an unexpected direction. Thus, the observer can easily switch between the total reflection illumination observation and the transmitted light observation without performing troublesome confirmation work.

  The present invention also provides an objective lens disposed opposite to the specimen, and illumination light consisting of substantially parallel light is incident on the entrance pupil position of the objective lens, and the illumination light is incident on the specimen by the objective lens. A scanning illumination optical system that condenses and scans the scanning light; a total reflection illumination optical system that condenses the illumination light off the axis of the entrance pupil position of the objective lens and causes evanescent light to enter the sample; and Incident switching means for switching incidence of the illumination light to the objective lens via the reflective illumination optical system or the scanning illumination optical system, and transmission detection for detecting the illumination light scanned by the scanning illumination optical system and transmitted through the sample An optical system, an openable / closable cover member disposed on the opposite side of the objective lens with the sample interposed therebetween, and an open / close sensor that detects opening / closing of the cover member, When the open state of the member is detected, to provide a microscope apparatus for switching the incident illumination light to the objective lens due to the incident switching means on the incident via said scanning illumination optical system.

  According to the present invention, when the cover member is opened, the optical path is automatically switched to the scanning illumination optical system side by the incident switching means, so that the illumination light is not incident on the objective lens via the total reflection illumination optical system. The Further, it is possible to prevent inconvenience that the illumination light that has not been totally reflected is emitted in an unexpected direction. Thus, the observer can easily switch between the total reflection illumination observation and the transmitted light observation without performing troublesome confirmation work.

In the above invention, the shutter may be provided in the total reflection illumination optical system.
In this way, even when the illumination light from the same light source is incident on the scanning illumination optical system and the total reflection illumination optical system, there is a problem that the illumination light for total reflection illumination is emitted in an unexpected direction. It can be prevented in advance.

  In the above invention, an incident state sensor for detecting the state of the incident switching unit is provided, and it is detected that the shutter is switched to incident illumination light to the total reflection illumination optical system by the incident state sensor. When the open state of the cover member is detected by the open / close sensor, illumination light incident on the objective lens via the total reflection illumination optical system may be blocked.

  When it is detected by the incident state sensor that the state of the incident switching means is a state in which illumination light is incident on the total reflection illumination optical system, illumination light consisting of substantially parallel light is emitted from the objective lens. If the cover member is opened carelessly, the illumination light may be emitted in an unexpected direction. Therefore, in this case, the illumination light is blocked from entering the objective lens through the total reflection illumination optical system by the shutter according to the detection of the open state of the cover member by the open / close sensor, thereby preventing the emission in an unexpected direction. It is possible to prevent the illumination light from being blocked by opening and closing the cover during other observation methods.

  Further, in the above invention, the cover member is provided between the sample and the transmission detection optical system, and is provided with an opening / closing part that opens and closes a part of the cover member, and the illumination switching unit is configured to perform the illumination. There may be provided an opening / closing section switching means for switching the opening / closing section to an open state when the incident light is switched to the scanning illumination optical system.

In this way, when the incident light is switched to the scanning illumination optical system by the incident switching means, the opening / closing section is switched to the open state by the opening / closing section switching means, and the pupil of the objective lens is switched via the scanning illumination optical system. Illumination light incident as substantially parallel light is condensed on the sample. Then, the transmitted light of the illumination light transmitted through the specimen passes through the opened opening / closing part and is incident on the transmission detection optical system, so that the transmitted illumination observation can be performed.
In this case, the observer can switch the observation method without performing the opening / closing operation of the cover member, and can further improve the handleability.

  According to the present invention, it is possible to suppress distortion generated in the optical system and to easily switch between total reflection illumination observation and transmitted light observation without performing a troublesome confirmation work by the observer.

1 is an overall configuration diagram schematically illustrating a microscope apparatus according to an embodiment of the present invention. FIGS. 2A and 2B are circuit diagrams illustrating a shutter drive circuit of the microscope apparatus of FIG. 1, wherein (a) a cover is attached and (b) a cover is removed. FIG. 5 is a circuit diagram showing a modification of the shutter drive circuit of FIG. 2, wherein (a) a cover is attached and (b) a cover is removed. It is a whole block diagram explaining typically the modification of the microscope apparatus of FIG. 4 is a shutter drive circuit of the microscope apparatus of FIG. 4, in which (a) the total reflection illumination is selected and the cover is removed, (b) the total reflection illumination is selected and the cover is attached, (c FIG. 4 is a circuit diagram showing a state where transmitted illumination is selected and a cover is removed, and (d) a state where transmitted illumination is selected and a cover is attached. It is a whole block diagram explaining typically the other modification of the microscope apparatus of FIG. 6 is a shutter drive circuit of the microscope apparatus of FIG. 6, wherein (a) the total reflection illumination is selected and the cover is removed, and (b) the transmitted illumination is selected and the cover is removed. FIG. FIG. 7A is a circuit diagram of a shutter drive circuit in a state where a total reflection illumination of the microscope apparatus of FIG. 6 is selected and a cover is mounted, and FIG. 7B is a diagram illustrating a state of an opening / closing door of the cover. FIG. 7A is a circuit diagram of a shutter drive circuit in a state where transmitted illumination of the microscope apparatus of FIG. 6 is selected and a cover is mounted, and FIG. It is a whole block diagram explaining typically the other modification of the microscope apparatus of FIG.

A microscope apparatus 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the microscope apparatus 1 according to the present embodiment includes a laser light source 2, two optical fibers 3 and 4 for guiding laser light emitted from the laser light source 2, and the optical fiber. The total reflection illumination optical system 5 and the scanning illumination optical system 6 that respectively guide the laser beams emitted from the end portions 3 and 4 are inserted into and removed from the optical axis at the subsequent stage of these optical systems 5 and 6. , A mirror (incident switching means) 7 that allows laser light to pass through any one of the optical systems, a shutter 8 that blocks or opens the optical path downstream of the mirror 7, and is incident when the shutter 8 is opened. A microscope main body 9 that performs observation with laser light, a control unit 10 that controls these and generates an image from image information acquired by the microscope main body 9, a monitor 11 that displays the generated image, and an observation method Enter And an input unit 12. In the figure, reference numeral 13 denotes a coupling lens.

The total reflection illumination optical system 5 includes a collimating lens 14 that makes laser light emitted from the end of the optical fiber 3 substantially parallel light, and a scanner that moves the laser light made substantially parallel light in a direction crossing the optical axis. 15, two sets of relay lenses 16, 17, 18, 19, a movable prism 20 for adjusting the optical path length, a field stop 21, and a shutter 22 that blocks or opens the optical path. Reference numerals 23 and 24 in the figure are mirrors. The field stop 21 is disposed at a position optically conjugate with the sample A, and is moved in the direction of an arrow b intersecting the optical axis by a driving source (not shown) so that the aperture diameter can be changed. It has become.
The movable prism 20 is moved in the direction indicated by the arrow a in the figure to adjust the optical path length in the total reflection illumination optical system 5.

  The optical path length is adjusted so that the laser light that has passed through the total reflection illumination optical system 5 is condensed at an off-axis position at the entrance pupil position of an objective lens 35 of the microscope body 9 described later. Thereby, the laser light that has passed through the total reflection illumination optical system 5 is emitted from the objective lens 35 to the specimen A in a substantially parallel light state. The laser beam emitted from the objective lens forms a predetermined angle with respect to the specimen A by being focused at an off-axis position at the pupil position of the objective lens 35, and is at a boundary position between the specimen A and the cover glass. Irradiated and totally reflected at that position.

  Further, the incident position on the specimen A is adjusted by adjusting the position of the field stop 21, and the incident range on the specimen A is adjusted by adjusting the aperture diameter of the field stop 21. Furthermore, by adjusting the deflection angle of the laser beam by the scanner 15, the incident angle of the laser beam to the specimen A can be adjusted, and the total reflection illumination range can be changed in the depth direction.

  The scanning illumination optical system 6 includes a collimator lens 25 that makes laser light emitted from the end of the optical fiber 4 substantially parallel light, a scanner 26 that two-dimensionally scans laser light that is made substantially parallel light, and 1 A pair of relay lenses 27, 28, a dichroic mirror 29 for branching the fluorescence returning through the relay lenses 27, 28 and the scanner 26 from the optical path of the laser light, a condenser lens 30, and a position optically conjugate with the sample A Are provided with a confocal pinhole 31 and two photodetectors 32 and 33. Reference numeral 34 in the drawing denotes a dichroic mirror that branches the fluorescence transmitted through the confocal pinhole 31 for each wavelength.

  The laser light that has passed through the scanning illumination optical system 6 is set to be incident as substantially parallel light on the entrance pupil position of the objective lens 35 of the microscope body 9. As a result, the laser light transmitted through the scanning illumination optical system 6 is focused on the specimen A arranged at the focal position of the objective lens 35.

  The microscope main body 9 includes an objective lens 35 that condenses the laser light that has passed through the total reflection illumination optical system 5 or the scanning illumination optical system 6, a stage 36 that faces the upper side of the objective lens 35 and supports the specimen, and a specimen A Is provided with a transmission light detector 37 and an eyepiece optical system 38. A mirror 39 and a dichroic mirror 40 are alternatively detachably provided between the eyepiece optical system 38 and the objective lens 35. Reference numerals 41 and 42 are mirrors. On the stage 36, a cover (cover member) 43 is detachably disposed at a position covering the specimen A manually. The transmission photodetector 37 is, for example, a photomultiplier tube.

  When the mirror 39 is inserted, the laser light reflected by the mirror 39 is incident on the objective lens 35. On the other hand, when the dichroic mirror 40 is inserted, the laser light reflected by the dichroic mirror 40 is condensed on the specimen A by the objective lens 35, and the fluorescence generated in the specimen A and condensed by the objective lens 35 is collected. And is guided to the eyepiece optical system 38.

The cover 43 is provided with two switches (sensors) 44 and 45 that detect attachment or removal of the cover 43. The switches 44 and 45 are configured to operate mechanically exclusively. The switches 44 and 45 are arranged in a parallel circuit as shown in FIG. The shutter 44 arranged in the total reflection illumination optical system 5 is connected in series to the switch 44, and a parallel circuit of the two switches 44 and 45 is provided between the optical systems 5 and 6 and the microscope body 9. The arranged shutters 8 are connected in series.
Each of the shutters 8 and 22 is configured to open the optical path when energized and to block the optical path when energization is stopped. Reference numeral 46 in the figure denotes a power switch.

The operation of the microscope apparatus 1 according to the present embodiment configured as described above will be described below.
In the preparation state of the specimen A, the power switch 46 is turned off, the energization to the shutters 8 and 22 is stopped, and the shutters 8 and 22 are closed, so that the laser light is incident on the microscope body 9. It has stopped.

  In this state, in order to perform total reflection illumination observation of the specimen A using the microscope apparatus 1 according to the present embodiment, the observer covers the specimen A on the specimen A mounted on the stage 36. The cover 43 is arranged and the power switch 46 is turned on. Since the switches 44 and 45 operate exclusively, the shutter 8 is energized and the shutter 8 is opened regardless of which switch 44 or 45 is closed. By disposing the cover 43, it is possible to prevent laser light incident from below the stage 36 from being transmitted above the cover 43.

  When the cover 43 is arranged, the two switches 44 and 45 are operated, and as shown in FIG. 2A, the switch 45 is opened, while the switch 44 is closed and the shutter 22 is energized, and the shutter 22 is opened. As a result, the laser beam can be incident through the total reflection illumination optical system 5. When total reflection illumination observation is input from the input unit 12 as an observation method, the mirror 7 is inserted on the optical path to form an optical path from the total reflection illumination optical system 5 to the microscope body 9. In the microscope main body 9, the dichroic mirror 40 is inserted on the optical path, and the observation by the eyepiece optical system 38 is selected.

  The laser light guided to the microscope body 9 via the total reflection illumination optical system 5 is reflected by the dichroic mirror 40, and then condensed at the entrance pupil position of the objective lens 35, and is applied to the specimen A from the tip of the objective lens 35. And emitted as parallel light having a predetermined angle with respect to the optical axis. Then, by being totally reflected at the boundary surface between the specimen A and the cover glass, evanescent light is incident on the specimen A, and the fluorescence generated in the specimen A is condensed by the objective lens 35 and transmitted through the dichroic mirror 40. The eyepiece optical system 38 can be visually observed.

  Further, when epi-illumination observation is input as an observation method from the input unit 12, the mirror 7 on the optical path is removed, and laser light via the scanning illumination optical system 6 is input to the microscope body 9. Become. In this case, in the microscope main body 9, a mirror 39 is arranged on the optical path instead of the dichroic mirror 40.

  The laser light guided to the microscope main body 9 via the scanning illumination optical system 6 is reflected by the mirror 39, then enters the entrance pupil position of the objective lens 35 as substantially parallel light, and is collected at the focal position of the objective lens 35. Shine. The fluorescence generated at the condensing position of the laser light is collected by the objective lens 35 and is returned to the laser by the dichroic mirror 29 provided in the scanning illumination optical system 6 while returning through the mirror 39 and the scanning illumination optical system 6. Only the light branched from the optical path of the light, condensed by the condensing lens 30 and passing through the confocal pinhole 31 is detected by the photodetectors 32 and 33. The condensing position of the laser beam on the specimen A is two-dimensionally scanned by the scanner 26. Therefore, the intensity of the fluorescence generated at each position and the scanning position by the scanner 26 are stored in association with each other. A dimensional fluorescence image can be generated.

Next, when the cover 43 is removed for some reason while the total reflection illumination observation is selected, the switch 44 is opened and the switch 45 is closed as shown in FIG. As a result, power to the shutter 22 is lost, and the shutter 22 is closed. Since the shutter 22 is disposed in the total reflection illumination optical system 5, the laser light through the total reflection illumination optical system 5 is prevented from entering the microscope body 9, and the objective lens can be removed even if the cover 43 is removed. Thus, it is possible to prevent laser light in a substantially parallel light state from being emitted from 35 in an unexpected direction.
In addition, when the cover 43 is removed for some reason while the epi-illumination is selected, the epi-illumination can be continuously observed without any particular change.

Next, when performing transmission illumination observation using the microscope apparatus 1 according to the present embodiment, the cover 43 is removed, and transmission illumination observation is input from the input unit 12 as an observation method.
In this case, since the shutter 22 is closed in the state of FIG. 2B, the laser light is incident on the specimen A as in the case of epi-illumination observation, but the transmission observation image sandwiches the specimen A. By storing the intensity of the laser light detected by the transmission light detector 37 disposed on the side opposite to the objective lens 35 and the scanning position by the scanner 26, a two-dimensional transmitted light image is stored. Can be generated.

  Thus, according to the microscope apparatus 1 according to the present embodiment, when removing the cover 43 from the stage 36, it is not necessary for the observer to confirm that total reflection illumination observation is not selected, and bothersome confirmation is made. The cover 43 can be freely removed without performing work. Further, in the state where the cover 43 is not disposed, even if total reflection illumination observation is input from the input unit 12 as an observation method, incidence of laser light through the total reflection illumination optical system 5 on the microscope body 9 is blocked. Is done. Therefore, even if the arrangement of the cover 43 is forgotten due to the carelessness of the observer, there is an advantage that it is possible to reliably prevent the laser light in a substantially parallel light state from being irradiated in an unexpected direction. Have.

In the present embodiment, the shutter 8 that opens and closes the optical path is provided on the optical path from the total reflection illumination optical system 5 and the scanning illumination optical system 6 to the microscope main body 9. As shown in FIG. 3, the shutter 8 may not be provided. 3A shows a state where the shutter 22 is opened, and FIG. 3B shows a state where the shutter 22 is closed.
In this way, the sample A can be exchanged during total reflection illumination observation, and the laser beam can be prevented from being emitted in an unexpected direction when switching from transmission illumination observation to total reflection illumination observation. be able to.

  Further, as shown in FIG. 4, an insertion / removal sensor 47 for detecting insertion / removal of the mirror 7 into / from the optical path is provided, and the mirror 7 is inserted into the optical path by the insertion / removal sensor 47. May be detected, the shutter 8 may be shut off in accordance with the open / closed state of the switches 44 and 45 that detect attachment / detachment of the cover 43. By doing in this way, as shown in FIG. 5, the incidence of the laser beam on the microscope body 9 can be blocked only when the cover 43 is removed for some reason during the total reflection illumination observation.

  That is, FIG. 5A shows a state in which the cover 44 is not disposed and the switch 44 is opened even though the total reflection illumination observation is selected and the insertion / removal sensor 47 is in the OFF state. Is shown. In this state, the shutter 8 is not energized, the shutter 8 is kept closed, and the incidence of laser light on the microscope body 9 is blocked.

  FIG. 5B shows a state in which total reflection illumination observation is selected and the cover 43 is arranged. In this state, when the switch 44 is closed, the shutter 8 is opened, and the laser light guided through the total reflection illumination optical system 5 is incident on the microscope main body 9 to perform total reflection illumination observation.

  Next, FIG. 5C shows a state in which transmitted illumination observation is selected, the insertion / removal sensor 47 is in the ON state, and the cover 43 is removed. In this state, the shutter 8 is energized through the insertion / removal sensor 47 in the ON state, and the laser light guided through the scanning illumination optical system 6 is incident on the microscope body 9 to perform transmission illumination observation. .

  On the other hand, FIG. 5D shows a state in which the cover 43 is arranged even though the transmitted illumination observation is selected. In this state, since the shutter 8 is energized through the ON / OFF sensor 47 and the closed switch 44, the shutter 8 is maintained in the open state, and the laser light microscope through the scanning illumination optical system 6 is used. Incidence to the main body 9 is performed, and fallen illumination observation can be performed.

In the above description, the case where the observer removes or installs the cover 43 has been described as an example. However, the present invention is not limited to this, and as shown in FIG. Alternatively, a door provided with an opening / closing door 43a that is opened and closed by a driving means (not shown) may be employed. The open / close door 43a is set to be closed when the insertion / removal sensor 47 is turned off and to be opened when the insertion / removal sensor 47 is turned on.
In this way, the removal of the cover 43 is limited to the time when the specimen A is set on the stage 36, etc. Depending on the observation method, the observer may not have to remove the cover 43, thereby improving handling. Can be improved.

In this case, as shown in FIGS. 7A and 7B, the cover 43 is removed, and as shown in FIGS. 8 and 9, the cover 43 is arranged. The operation is different.
When the load resistance R is connected in parallel to the open / close door 43a and the insertion / removal sensor 47 is turned on, the shutter 8 is energized through the load resistance R even if the cover 43 is not disposed. It has become.

  That is, FIG. 7A shows a state in which the cover 44 is not disposed and the switch 44 is opened even though the total reflection illumination observation is selected and the insertion / removal sensor 47 is in the OFF state. Is shown. In this state, the shutter 8 is not energized, the shutter 8 is kept closed, and the incidence of laser light on the microscope body 9 is blocked.

  FIG. 7B shows a state in which transmitted illumination observation is selected, the insertion / removal sensor 47 is in the ON state, and the cover 43 is removed. In this state, the shutter 8 is energized through the insertion / removal sensor 47 and the load resistance R in the ON state, and the laser light guided through the scanning illumination optical system 6 is incident on the microscope main body 9 to transmit illumination. Observations can be made.

  Further, FIG. 8A shows that the total reflection illumination observation is selected and the insertion / removal sensor 47 is in the OFF state, and the cover 43 is attached, corresponding to the OFF state of the insertion / removal sensor 47, As shown in FIG. 8B, a state in which the open / close door 43a of the cover 43 is closed is shown. When the switch 44 is closed, the shutter 8 is opened, and the laser light guided through the total reflection illumination optical system 5 is incident on the microscope main body 9 so that total reflection illumination observation can be performed.

Further, FIG. 9A shows that the transmitted illumination observation is selected and the insertion / removal sensor 47 is in the ON state, and the cover 43 is attached, corresponding to the ON state of the insertion / removal sensor 47. 9 (b) shows a state in which the open / close door 43a of the cover 43 is opened. When the switch 44 is closed, the shutter 8 is opened, laser light is incident on the microscope body 9 via the scanning illumination optical system 6, and transmitted illumination is performed by the transmission light detector 37 via the open / close door 43a. Observations can be made.
Further, the controller 10 may open and close the opening / closing door 43a based on a signal from the insertion / removal sensor 47 without directly interlocking the insertion / removal sensor 47 and the opening / closing door 43a.

  Further, as shown in FIG. 10, the optical paths in the total reflection illumination optical system 5 may be switched by disposing the mirrors 23 and 24 so as to be insertable into and removable from the optical path. In the figure, reference numerals 48 and 49 are sensors for detecting insertion / removal of the mirrors 23 and 24, and reference numerals 50 and 51 are shutters.

  When the mirrors 23 and 24 are inserted, an optical path for total reflection illumination can be generated, and when the mirrors 23 and 24 are removed, another optical path for laser scanning illumination can be generated. In this case, each optical path is opened and closed by the shutters 22 and 50 according to the output signals of the sensors 48 and 49 that detect insertion / removal of the mirrors 23 and 24 and the state of the switch 44 that detects attachment / detachment of the cover 43. do it.

  Further, when the total reflection illumination observation is selected and the mirror 7 is inserted on the optical path, the optical path is blocked by the shutter 51 disposed in the scanning illumination optical system 6, whereby the scanning illumination optical system 6. Incident laser light is reliably prevented.

In the above embodiment, the shutters 8, 22, 50, 51 are not limited to mechanical ones that are inserted into and removed from the optical path, and an acousto-optic element may be employed, or the laser light source 2 may be used. Optical quenching means such as dimming in itself or an interferometer may be employed.
In the above embodiment, the shutters 8, 22, 50, 51 and the sensors 44, 45, 47, 48, 49 are interlocked. Instead, the sensors 44, 45, 47, 48, 49 are used. The control unit 10 may open and close the shutters 8, 22, 50, 51 based on the signal from.

Further, the total reflection light is blocked by the shutters 22 and 50. Instead, the optical path is switched to the scanning illumination optical system 6 side by the mirror 7 so that the total reflection light does not enter the objective lens 35. It may be.
Further, as the incident switching means, the mirror 7 that is inserted into and removed from the optical path and switches the optical path of the total reflection illumination optical system 5 and the scanning illumination optical system 6 is illustrated. A dichroic mirror that allows the laser light from the illumination optical system 5 and the scanning verification optical system 6 to enter the same optical path may be employed, and the incident switching means may be configured by the shutters 22, 50, and 51.

A Specimen 1 Microscope device 5 Total reflection illumination optical system 6 Scanning illumination optical system 7 Mirror (incident switching means)
8 Shutter 22, 50, 51 Shutter (shutter, incident switching means)
35 Objective lens 37 Photodetector for transmission (detection optical system for transmission)
43 Cover (cover member)
43a Open / close door
44, 45 switch (open / close sensor)
47 Insertion / removal sensor (incident state sensor: switching part)

Claims (5)

An objective lens placed opposite the specimen;
A scanning illumination optical system that causes illumination light consisting of substantially parallel light to enter the entrance pupil position of the objective lens, and that causes the illumination light to be condensed and scanned on the sample by the objective lens;
A total reflection illumination optical system that condenses the illumination light off-axis of the entrance pupil position of the objective lens and makes the specimen enter evanescent light;
Incident switching means for switching incidence of the illumination light to the objective lens via the total reflection illumination optical system or the scanning illumination optical system;
A transmission detection optical system that detects illumination light scanned by the scanning illumination optical system and transmitted through the specimen;
An openable and closable cover member disposed on the opposite side of the objective lens across the specimen;
An open / close sensor for detecting opening and closing of the cover member;
A microscope apparatus comprising: a shutter that blocks illumination light from entering the objective lens through the total reflection illumination optical system when the open / close sensor detects the open state of the cover member. An objective lens placed opposite the specimen;
A scanning illumination optical system that causes illumination light consisting of substantially parallel light to enter the entrance pupil position of the objective lens, and that causes the illumination light to be condensed and scanned on the sample by the objective lens;
A total reflection illumination optical system that condenses the illumination light off-axis of the entrance pupil position of the objective lens and makes the specimen enter evanescent light;
Incident switching means for switching incidence of the illumination light to the objective lens via the total reflection illumination optical system or the scanning illumination optical system;
A transmission detection optical system that detects illumination light scanned by the scanning illumination optical system and transmitted through the specimen;
An openable and closable cover member disposed on the opposite side of the objective lens across the specimen;
An opening / closing sensor for detecting opening / closing of the cover member,
A microscope apparatus that switches the incidence of illumination light on the objective lens by the incidence switching means to incidence via the scanning illumination optical system when the open / close sensor detects the open state of the cover member. An incident state sensor for detecting the state of the incident switching means;
When the open state of the cover member is detected by the open / close sensor when it is detected that the shutter is switched to the incidence of illumination light to the total reflection illumination optical system by the incident state sensor The microscope apparatus according to claim 1, wherein illumination light entering the objective lens through the total reflection illumination optical system is blocked . The cover member is disposed between the specimen and the transmission detection optical system, and is provided with an opening / closing part that opens and closes a part of the cover member,
The open / close unit switching unit that switches the open / close unit to an open state when the incident light is switched to the scanning illumination optical system by the incident switching unit. microscope apparatus according to any. The microscope apparatus according to claim 1 , wherein the shutter is provided in the total reflection illumination optical system .

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