JP6355928B2 - Fluorescence observation equipment - Google Patents

Description

  The present invention relates to a fluorescence observation apparatus.

  Conventionally, there has been known a fluorescence observation apparatus that performs fluorescence observation of a specimen using evanescent light that oozes out from the cover glass by totally reflecting laser light at the boundary surface between the cover glass and the specimen (for example, Patent Document 1 and (See Patent Document 2).

  The fluorescence observation apparatus described in Patent Document 1 deflects laser light emitted from a light source with a galvanometer, thereby shifting the condensing position of the laser light at the pupil position of the objective lens from the center of the pupil and totally reflecting the laser light. And evanescent light is generated. Further, the fluorescence observation apparatus described in Patent Document 2 moves the emission position of the optical fiber that guides the laser beam, thereby shifting the laser beam condensing position at the pupil position of the objective lens from the center of the pupil. The light is reflected to generate evanescent light.

Japanese Patent No. 4934281 Japanese Patent No. 4563699

  However, in the fluorescence observation apparatuses of Patent Documents 1 and 2, since the laser beam condensing position can be moved only in one direction at the pupil position of the objective lens, only evanescent light that oozes from one direction is irradiated to the specimen. Can not do it. For this reason, there is an inconvenience that the irradiation of the evanescent light is biased and the specimen cannot be fluorescently observed with high accuracy.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a fluorescence observation apparatus that can evenly irradiate a specimen with evanescent light and accurately observe the specimen with fluorescence.

In order to achieve the above object, the present invention provides the following means.
The present invention includes an objective lens disposed opposite to a specimen, a relay optical system that condenses illumination light emitted from a light source at a pupil position of the objective lens, and an optical conjugate with a focal position of the objective lens. A phase modulator that is arranged at a different position and capable of modulating the phase of the illumination light from the light source, and the sample is obtained by condensing the illumination light at a position shifted from the center position of the pupil at the pupil position by the phase modulator And a control unit that sets a predetermined phase modulation pattern to irradiate evanescent light.

  According to the present invention, the illumination light emitted from the light source is phase-modulated by the phase modulation unit based on the predetermined phase modulation pattern, and is shifted from the center position of the pupil in the pupil position of the objective lens by the relay optical system. By condensing, the illumination light is totally reflected on the specimen and irradiated with evanescent light. Thereby, fluorescence observation with a very dark background and high contrast can be performed based on the return light that returns from the local region where the evanescent light reaches the sample.

  In this case, since the condensing position of the illumination light at the pupil position of the objective lens is determined by a predetermined phase modulation pattern of the phase modulation unit, the control unit generates evanescent light from the periphery substantially evenly with respect to the sample. If a phase modulation pattern for condensing illumination light is set on the specimen, the specimen can be irradiated with evanescent light without bias, and the specimen can be accurately observed with fluorescence.

In the above invention, the control unit may set the predetermined phase modulation pattern for condensing illumination light at a desired position in a direction intersecting the optical axis at the pupil position.
With this configuration, evanescent light can be generated from a desired direction with respect to the specimen. Therefore, the specimen can be irradiated with evanescent light from a desired direction without deviation, and the specimen can be fluorescently observed with high accuracy.

In the above invention, the control unit may set the predetermined phase modulation pattern for simultaneously condensing illumination light at a plurality of positions in a direction intersecting the optical axis at the pupil position.
With this configuration, it is possible to generate evanescent light from a plurality of directions with respect to the specimen. Therefore, the specimen can be irradiated with evanescent light from a plurality of directions without deviation, and the specimen can be fluorescently observed with high accuracy.

In the above invention, the control unit may set the predetermined phase modulation pattern for condensing illumination light in an annular shape so as to surround the optical axis at the pupil position.
With this configuration, evanescent light can be generated from the entire circumferential direction with respect to the sample. Therefore, the specimen can be irradiated with evanescent light from all over the circumferential direction without deviation, and the specimen can be fluorescently observed with high accuracy.

In the above invention, the control unit may adjust the predetermined phase modulation pattern so as to correct a condensing position of illumination light in a direction orthogonal to and / or along an optical axis at the pupil position.
With this configuration, even when the condensing position in the direction orthogonal to or along the optical axis at the pupil position of the objective lens is shifted, the irradiation direction of the evanescent light on the specimen is corrected and fluorescence observation is performed with high accuracy. be able to.

  In the above-mentioned invention, the control unit is configured to collect the illumination light by the aberration of the lens disposed in the optical path from the light source to the objective lens and / or the disturbance of the wave front due to the surface accuracy of the reflecting member in the optical path. The phase modulation pattern may be adjusted to correct the deterioration.

  The control unit corrects the deterioration of the collection performance of the illumination light due to the aberration of the relay optical system arranged in the optical path from the light source to the objective lens and the disturbance of the wavefront due to the surface accuracy of the reflecting member, thereby improving the efficiency of the illumination light. The specimen can be fully reflected and irradiated with evanescent light with less noise to the specimen, so that the specimen can be observed with fluorescence with high accuracy.

  In the invention described above, the light source capable of generating illumination light having different wavelengths is provided, and the control unit is configured to perform the predetermined phase modulation so as to correct a deviation of a condensing position according to a wavelength of illumination light emitted from the light source. The pattern may be adjusted.

  When the wavelength of the illumination light changes, the condensing position may shift due to chromatic aberration of the illumination light. By configuring in this way, the illumination light can be condensed at a desired position without any misalignment even when illuminating any illumination light while changing the illumination light to an appropriate wavelength according to the fluorescent substance of the sample. Efficient total reflection can be achieved. Thereby, the specimen can be irradiated with evanescent light with less noise, and the specimen can be observed with fluorescence with high accuracy.

  In the above invention, the predetermined lens unit includes a plurality of objective lenses having different pupil positions, and the control unit corrects the deviation of the condensing position according to the pupil position of the objective lens disposed in the optical path of the illumination light. It is also possible to adjust the phase modulation pattern.

  With this configuration, while changing to an objective lens with an appropriate magnification according to the sample, the illumination light can be condensed at a desired position without any positional deviation and total reflection can be efficiently performed regardless of the objective lens used. Can be made. Thereby, the specimen can be irradiated with evanescent light with less noise, and the specimen can be observed with fluorescence with high accuracy.

  In the above invention, the illumination light is reflected or transmitted with different wavelength characteristics, and the return light returning from the sample is transmitted or reflected by the illumination light and branched from the optical path of the illumination light. A plurality of branching units may be provided, and the control unit may adjust the predetermined phase modulation pattern so as to correct a deviation of a condensing position according to the branching unit arranged in the optical path of the illumination light.

  With this configuration, while changing to an appropriate branching unit according to the wavelength of the return light to be detected, the illumination light can be condensed at a desired position without any positional deviation even when any branching unit is used. Can be totally totally reflected. Thereby, the specimen can be irradiated with evanescent light with less noise, and the specimen can be observed with fluorescence with high accuracy.

  In the above invention, a temperature measuring unit that measures a temperature around the sample is provided, and the control unit corrects the deviation of the condensing position according to the temperature measured by the temperature measuring unit. The modulation pattern may be adjusted.

  With such a configuration, even when the temperature around the sample changes, the illumination light can be condensed at a desired position without being displaced and can be efficiently totally reflected. Thereby, the specimen can be irradiated with evanescent light with less noise, and the specimen can be observed with fluorescence with high accuracy.

  According to the present invention, there is an effect that the specimen can be irradiated with evanescent light without any deviation and the specimen can be accurately observed with fluorescence.

It is a schematic block diagram which shows the fluorescence observation apparatus which concerns on 1st Embodiment of this invention. It is a figure which shows an example of the condensing position of the laser beam in the pupil position of the objective lens in the case of performing total reflection illumination observation with the fluorescence observation apparatus of FIG. It is a figure which shows an example of the condensing position of the laser beam in the pupil position of the objective lens in the case of carrying out total reflection illumination observation with the conventional fluorescence observation apparatus as a comparative example of the fluorescence observation apparatus which concerns on 1st Embodiment of this invention. It is a schematic block diagram which shows the fluorescence observation apparatus which concerns on 2nd Embodiment of this invention.

[First Embodiment]
A fluorescence observation apparatus according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the fluorescence observation apparatus 100 according to the present embodiment has a light source 1 that generates laser light (illumination light) and a laser beam emitted from the light source 1 with a predetermined modulation pattern (phase modulation pattern). LCOS-SLM (Liquid Crystal on Silicon-Spatial Light Modulator, spatial light phase modulator, phase modulation unit) 5 that performs phase modulation based on the above, a control unit 7 that sets a predetermined modulation pattern of the LCOS-SLM 5, and laser light The relay optical system 11 that relays the laser beam, the cut filter 13 that cuts the 0th-order light from the laser light relayed by the relay optical system 11, and the laser light that is disposed facing the sample S and relayed by the relay optical system 11 The objective which collects the return light including the fluorescence generated in the specimen S And a lens 17.

Reference numeral 3 denotes a reflection mirror (reflecting member) that reflects the laser light from the light source 1 toward the LCOS-SLM 5, and reference numeral 9 denotes a reflection that reflects the laser light from the LCOS-SLM 5 toward the relay optical system 11. A mirror (reflecting member) is shown. Symbol E indicates the pupil position of the objective lens 17.
The sample S is stained with a fluorescent dye and placed on a glass plate 15 such as a slide glass.

  The fluorescence observation apparatus 100 also includes a dichroic mirror 19 that branches the return light collected by the objective lens 17 from the optical path of the laser light, and reflected light of the laser light among the return lights branched from the optical path of the laser light. And a CCD (Charge-Coupled Device) 27 that acquires an image of the specimen S are provided. Reference numeral 23 denotes an imaging lens that forms an image of the fluorescence that has passed through the barrier filter 21 on the imaging surface of the CCD 27, and reference numeral 25 denotes a reflection mirror.

  The LCOS-SLM 5 includes a large number of micro-modulation elements (not shown) arranged two-dimensionally so that the amount of phase change given to the incident light can be individually controlled for each micro-modulation element. It has become. The LCOS-SLM 5 is disposed at a position optically conjugate with the focal position of the objective lens 17. The LCOS-SLM 5 is configured to change the wavefront shape of the incident laser light by phase modulation for each minute modulation element based on a predetermined modulation pattern set by the control unit 7 and emit it.

  For example, the LCOS-SLM 5 moves the condensing position of the laser light at the pupil position E of the objective lens 17 in a direction intersecting the optical axis according to a predetermined modulation pattern, or a plurality of positions at the pupil position E of the objective lens 17. A laser beam can be simultaneously focused on the location.

  In the case of total reflection illumination observation, the control unit 7 causes the LCOS-SLM 5 to focus the laser beam at a position shifted from the center of the pupil at the pupil position E of the objective lens 17 as shown in FIG. A modulation pattern for leaching evanescent light from 15 is set. In the present embodiment, as shown in FIG. 2, four desired positions spaced apart from each other by an equal distance from the center of the pupil at the pupil position E of the objective lens 17 in the direction orthogonal to the optical axis and spaced equally in the circumferential direction. An example of a pattern for simultaneously condensing laser light will be described. In FIG. 2, the condensing position of the laser beam at the pupil position E of the objective lens 17 is indicated by a symbol K. The same applies to FIG.

  The laser light is obliquely incident on the glass plate 15 of the sample S by shifting the condensing position of the laser light from the center of the pupil at the pupil position E of the objective lens 17 in a direction orthogonal to the optical axis by the LCOS-SLM 5. Then, the sample S is irradiated with evanescent light that is totally reflected in the glass plate 15 and exudes to the sample S side from the sample side interface of the glass plate 15.

  The dichroic mirror 19 reflects the laser light relayed by the relay optical system 11 toward the objective lens 17, and transmits the return light collected by the objective lens 17 and returning the optical path of the laser light to the barrier filter 21. It is made to enter.

The operation of the fluorescence observation apparatus 100 configured as described above will be described.
When the specimen S is observed with total reflection illumination by the fluorescence observation apparatus 100 according to this embodiment, a modulation pattern for total reflection illumination by the control unit 7 is set in the LCOS-SLM 5 and laser light is generated from the light source 1.

  As shown in FIG. 1, the laser light emitted from the light source 1 is phase-modulated and reflected by the LCOS-SLM 5 via the reflection mirror 3, and then reflected by the reflection mirror 9, the relay optical system 11, the cut filter 13, and the dichroic. The light is condensed at the pupil position E of the objective lens 17 through the mirror 19. The zero-order light collected at the center of the pupil position E of the objective lens 17 is cut by the cut filter 13.

  In this case, the laser beams are separated from each other by an equal distance in the direction perpendicular to the optical axis from the center of the pupil at the pupil position E of the objective lens 17 according to the modulation pattern of the LCOS-SLM 5 as shown in FIG. The light is condensed simultaneously at four desired locations (see symbol K in FIG. 2) that are equally spaced in the circumferential direction. Then, the laser light is obliquely incident on the glass plate 15 of the sample S from the objective lens 17 and is totally reflected at the interface between the glass plate 15 and the sample S, and the evanescent light that exudes from the glass plate 15 is locally applied to the sample S. Irradiated.

  The fluorescence generated in the sample S by locally irradiating the sample with evanescent light is photographed by the CCD 27 via the objective lens 17, the dichroic mirror 19, the imaging lens 23 and the reflection mirror 25. As a result, the CCD 27 can acquire a fluorescent image of a very shallow region where the evanescent light in the sample S has reached, and can perform fluorescence observation with a very dark background and high contrast.

  In this case, since the condensing position of the laser light at the pupil position E of the objective lens 17 is determined by a predetermined modulation pattern of the LCOS-SLM 5, the control unit 7 emits evanescent light from the periphery substantially evenly with respect to the sample S. By setting a modulation pattern for condensing the laser light so as to be generated, the evanescent light can be applied to the specimen S without any deviation.

  In the present embodiment, as shown in FIG. 2, four desired positions spaced apart from each other by an equal distance from the center of the pupil at the pupil position E of the objective lens 17 in the direction orthogonal to the optical axis and spaced equally in the circumferential direction. (See symbol K in FIG. 2) By condensing the laser beam simultaneously, the evanescent light can be applied to the specimen S from the surrounding four locations without any deviation.

  As a comparative example of the fluorescence observation apparatus 100 according to the present embodiment, the laser light is deflected by a galvanometer as in the conventional fluorescence observation apparatus, and the condensing position of the laser light at the pupil position of the objective lens is changed from the center of the pupil. In the configuration of shifting, as shown in FIG. 3, the condensing position of the laser beam (see symbol K in FIG. 3) can be moved only in one direction at the pupil position of the objective lens. Therefore, only evanescent light that oozes out from one direction can be irradiated on the specimen, and the irradiation of the evanescent light is biased.

  As described above, according to the fluorescence observation apparatus 100 according to the present embodiment, the modulation pattern for condensing the laser light by the control unit 7 so that the evanescent light is generated from the periphery substantially evenly with respect to the specimen S. By setting, the specimen S can be irradiated with evanescent light without deviation, and the specimen S can be accurately observed with fluorescence.

This embodiment can be modified as follows.
That is, in the present embodiment, the control unit 7 sets a modulation pattern for condensing laser light at a plurality of positions shifted in the direction orthogonal to the optical axis from the center of the pupil at the pupil position E of the objective lens 17. It was decided. As a modified example, the control unit 7 may set a modulation pattern that simultaneously collects laser beams in an annular shape so as to surround the optical axis at the pupil position E of the objective lens 17.

  In this way, the LCOS-SLM 5 simultaneously collects the laser light in an annular shape surrounding the optical axis at the pupil position E of the objective lens 17 and totally reflects it within the glass plate 15, and circumferentially with respect to the sample S. The evanescent light can be generated from the entire area. Therefore, the specimen S can be irradiated with evanescent light from the entire circumferential direction without deviation, and the specimen S can be fluorescently observed with higher accuracy.

[Second Embodiment]
Next, a fluorescence observation apparatus according to the second embodiment of the present invention will be described.
As shown in FIG. 4, the fluorescence observation apparatus 200 according to the present embodiment is different from the light source 1, the objective lens 17, and the dichroic mirror 19 in that the pupil position E is different from the light source 101 that can generate laser light having different wavelengths. A plurality of objective lenses 17A, 17B, a plurality of dichroic mirrors (branching units) 19A, 19B having different wavelength characteristics, and a temperature measuring device (temperature measuring unit) 129 for measuring the temperature around the sample S, The control unit 7 is different from the first embodiment in that the control unit 7 adjusts the predetermined modulation pattern of the set LCOS-SLM 5.
In the following, portions having the same configuration as those of the fluorescence observation apparatus 100 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  The light source 101 includes a first light source unit 31A that generates a 635 nm laser beam, a second light source unit 31B that generates a 515 nm laser beam, a third light source unit 31C that generates a 488 nm laser beam, and a 405 nm laser beam. And a fourth light source unit 31D.

  The light source 101 also includes reflection mirrors 33 and 41 and dichroic mirrors 35, 37, and 39 that combine optical paths of laser light emitted from the light source units 31A, 31B, 31C, and 31D, and the wavelength of the emitted laser light. An AOTF (Acousto Optical Tunable Filter) 43 for switching is provided.

  The reflection mirror 33 reflects the laser light emitted from the first light source unit 31 </ b> A toward the dichroic mirror 35. The dichroic mirror 35 transmits the laser light incident from the first light source unit 31A via the reflection mirror 33, while reflecting the laser light emitted from the second light source unit 31B to synthesize these optical paths. It has become. The dichroic mirror 37 transmits the laser light from the first light source unit 31A and the second light source unit 31B, while reflecting the laser light from the third light source unit 31C to synthesize the optical paths of these laser light. It has become. The dichroic mirror 39 combines the laser light from the first light source unit 31A to the third light source unit 31C, and reflects the laser light from the fourth light source unit 31D to combine these optical paths. Yes. The reflection mirror 41 reflects the laser light from the first light source unit 31 </ b> A to the fourth light source unit 31 </ b> D combined with the optical path by the dichroic mirror 39 toward the AOTF 43.

The plurality of objective lenses 17A and 17B are supported by the revolver 18 so as to be selectively arranged on the optical path of the laser light.
The plurality of dichroic mirrors 19 </ b> A and 19 </ b> B are held by the turret 20 together with the barrier filter 21. A set of these dichroic mirrors 19A and 19B and the barrier filter 21 can be selectively arranged on the optical path of the return light by the turret 20.

  The controller 7 is disposed between the light source 101 and the objective lens 17 such as the aberration of the relay optical system 11 and the surface accuracy of the reflecting mirrors 3 and 9 after setting a predetermined modulation pattern of the LCOS-SLM 5. In accordance with the aberration of the lens and the surface accuracy of the reflecting member, the modulation pattern is adjusted so as to correct the deterioration of the laser beam condensing performance (spreading of the condensing spot) due to the wavefront disturbance according to the aberration and surface accuracy. It has become. The control unit 7 adjusts the modulation pattern according to the wavelength of the laser light emitted from the light source 101 so as to correct the deviation of the condensing position of the laser light according to the wavelength. Further, the control unit 7 adjusts the modulation pattern according to the pupil position E of the objective lenses 17A and 17B arranged in the optical path of the laser light so as to correct the deviation of the condensing position of the laser light according to the pupil position E. It is like that. Further, the control unit 7 adjusts the modulation pattern in accordance with the wavelength characteristics of the dichroic mirrors 19A and 19B arranged in the optical path of the laser light so as to correct the deviation of the condensing position of the laser light according to the wavelength characteristics. It has become. Further, the control unit 7 adjusts the modulation pattern so as to correct the deviation of the condensing position of the laser light according to the temperature change around the sample S according to the temperature measured by the temperature measuring device 129. Yes.

The operation of the fluorescence observation apparatus 200 configured as described above will be described.
When the specimen S is observed with total reflection illumination by the fluorescence observation apparatus 200 according to the present embodiment, a predetermined modulation pattern for total reflection illumination by the control unit 7 is set in the LCOS-SLM 5 and any one of the objective lenses 17A is set by the revolver 18. , 17B are arranged on the optical path, and one of the dichroic mirrors 19A, 19B is arranged on the optical path by the turret 20.

  Next, in the light source 101, the laser light generated from any of the light source units 31A, 31B, 31C, and 31D is emitted by the AOTF 43. Laser light emitted from the light source 101 is reflected by the dichroic mirror 19A (19B) on the optical path via the reflection mirror 3, the LCOS-SLM 5, the reflection mirror 9, the relay optical system 11, and the cut filter 13.

  Then, the laser light is condensed at a position shifted in the direction perpendicular to the optical axis from the center of the pupil at the pupil position E of the objective lens 17A (17B) on the optical path in accordance with the modulation pattern of the LCOS-SLM5. It is totally reflected at the interface with the glass plate 15. As a result, the evanescent light that exudes from the glass plate 15 is locally irradiated on the specimen S.

  The fluorescence generated in the specimen S by irradiation with the evanescent light passes through the objective lens 17A (17B) on the optical path, the dichroic mirror 19A (19B) on the optical path, the barrier filter 21, the imaging lens 23, and the reflection mirror 25. The image is taken by the CCD 27. As a result, a fluorescent image of a very shallow region where the evanescent light in the sample S reaches the CCD 27 is acquired.

  In this case, the laser beam is caused by the aberration of the lens disposed between the light source 101 and the objective lens 17 such as the aberration of the relay optical system 11 and the surface accuracy of the reflecting mirrors 3 and 9 and the disturbance of the wavefront due to the surface accuracy of the reflecting member. The focusing performance of the objective lens 17A (17B) disposed in the optical path of the laser beam is shifted to the pupil position E of the objective lens 17A (17B) disposed in the optical path of the laser beam. Accordingly, the laser light condensing position is shifted, the laser light converging position is shifted according to the dichroic mirror 19A (19B) arranged in the optical path of the laser light, or around the specimen S. If the laser beam condensing position shifts due to temperature change, the controller 7 changes the LCOS-SLM 5 so as to correct the deterioration of the laser beam condensing performance and the condensing position shift. Pattern is adjusted.

  Therefore, according to the fluorescence observation apparatus 200 according to the present embodiment, when the specimen S is observed with total reflection illumination, the control unit 7 causes the wavefront disturbance due to the aberration of the relay optical system 11 and the surface accuracy of the reflection mirrors 3 and 9. It is possible to eliminate the degradation of the laser beam condensing performance due to the laser beam and efficiently totally reflect the laser beam.

  Further, while changing to laser light of an appropriate wavelength according to the fluorescent material of the sample S by the light source 101, the laser light is condensed at a desired position without any positional deviation even when irradiating any laser light. It can be totally reflected. In addition, while changing to the objective lenses 17A and 17B having an appropriate magnification according to the sample S, the laser light is condensed at a desired position without any positional deviation, and any of the objective lenses 17A and 17B is used efficiently. Can be reflected. Further, while changing to the dichroic mirrors 19A and 19B having an appropriate wavelength characteristic in accordance with the wavelength of the return light to be detected, the laser light is condensed at a desired position without any positional deviation when using any of the dichroic mirrors 19A and 19B. Can be efficiently totally reflected. Furthermore, even when the temperature around the specimen S changes, the laser light can be condensed at a desired position without being displaced and efficiently totally reflected. As a result, the specimen S can be irradiated with evanescent light with less noise, and the specimen S can be fluorescently observed with high accuracy.

  The embodiment of the present invention has been described in detail with reference to the drawings. However, the specific configuration is not limited to this embodiment, and includes design changes and the like within a scope not departing from the gist of the present invention. For example, the present invention is not limited to those applied to each of the above embodiments, and may be applied to embodiments in which these embodiments are appropriately combined, and is not particularly limited.

  Further, in each of the above-described embodiments, the modulation pattern for condensing the laser beam simultaneously at a plurality of desired positions shifted from the center of the pupil at the pupil position E of the objective lens 17 in the direction intersecting the optical axis, or the objective lens 17. The modulation pattern for simultaneously condensing laser light in a ring shape so as to surround the optical axis at the pupil position E is described as an example, but the sample S is irradiated with evanescent light without deviation according to the phase modulation pattern of the LCOS-SLM5. However, the present invention is not limited to this.

  Moreover, you may provide the insertion / removal mechanism which inserts / removes the cut filter 13 with respect to the optical path of a laser beam. The cut filter 13 is removed from the optical path by the insertion / removal mechanism, and the laser light from the light sources 1 and 101 is condensed at the center of the pupil position E of the objective lens 17 by controlling so as not to perform the phase modulation by the LCOS-SLM 5. Normal epi-illumination observation can be performed.

1,101 Light source 3 Reflection mirror (reflection member)
5 LCOS-SLM (Phase Modulator)
7 Control unit 9 Reflection mirror (reflection member)
11 Relay optical system 17, 17A, 17B Objective lens 19A, 19B Dichroic mirror (branch part)
129 Temperature measuring device (Temperature measuring unit)
100,200 Fluorescence observation device E Pupil position S Sample

Claims (10)

An objective lens placed opposite the specimen;
A relay optical system for condensing the illumination light emitted from the light source at the pupil position of the objective lens;
A phase modulator arranged at a position optically conjugate with the focal position of the objective lens and capable of modulating the phase of illumination light from the light source;
Fluorescence observation apparatus comprising: a control unit that sets a predetermined phase modulation pattern so that illumination light is collected at a position shifted from the center position of the pupil at the pupil position by the phase modulation unit and the sample is irradiated with evanescent light .   The fluorescence observation apparatus according to claim 1, wherein the control unit sets the predetermined phase modulation pattern for condensing illumination light at a desired position in a direction intersecting the optical axis at the pupil position.   The fluorescence observation apparatus according to claim 1, wherein the control unit sets the predetermined phase modulation pattern for simultaneously condensing illumination light at a plurality of positions in a direction intersecting the optical axis at the pupil position.   The fluorescence observation apparatus according to claim 1, wherein the control unit sets the predetermined phase modulation pattern for condensing illumination light in an annular shape so as to surround an optical axis at the pupil position.   5. The control unit according to claim 1, wherein the control unit adjusts the predetermined phase modulation pattern so as to correct a condensing position of illumination light in a direction orthogonal to and / or along an optical axis at the pupil position. The fluorescence observation apparatus according to 1.   The control unit corrects the deterioration of the focusing performance of the illumination light due to the aberration of the lens disposed in the optical path from the light source to the objective lens and / or the disturbance of the wavefront due to the surface accuracy of the reflecting member in the optical path. The fluorescence observation apparatus according to claim 5, wherein the predetermined phase modulation pattern is adjusted. The light source capable of generating illumination light having different wavelengths,
The fluorescence observation apparatus according to claim 5, wherein the control unit adjusts the predetermined phase modulation pattern so as to correct a shift of a condensing position according to a wavelength of illumination light emitted from the light source. A plurality of objective lenses having different pupil positions;
The fluorescence observation apparatus according to claim 5, wherein the control unit adjusts the predetermined phase modulation pattern so as to correct a deviation of a condensing position according to a pupil position of the objective lens arranged in an optical path of illumination light. A plurality of branch portions having different wavelength characteristics and reflecting or transmitting illumination light, and transmitting or reflecting return light returning from the sample when irradiated with illumination light and branching from the optical path of the illumination light Prepared,
The fluorescence observation apparatus according to claim 5, wherein the control unit adjusts the predetermined phase modulation pattern so as to correct a deviation of a condensing position according to the branching unit disposed in the optical path of illumination light. A temperature measuring unit for measuring the ambient temperature of the sample;
The fluorescence observation apparatus according to claim 5, wherein the control unit adjusts the predetermined phase modulation pattern so as to correct a deviation of a condensing position according to a temperature measured by the temperature measurement unit.

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