JP2013231930A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope device that enables fine adjustment of intensity of a laser beam even when the laser beam is used with the intensity reduced, and also enables stable observation.SOLUTION: A microscope device 1 includes a light source unit 2 for outputting a laser beam, an observation optical system 3 for irradiating a sample with a laser beam output from the light source unit 2 to observe the sample, a fiber optical system 4 for guiding the laser beam from the light source unit 2 to the observation optical system 3, and two modulation means 5, 6 disposed in series on an optical path from the light source unit 2 to the observation optical system 3 for modulating the intensity of the laser beam.

Description

  The present invention relates to a microscope apparatus.

  2. Description of the Related Art Conventionally, a microscope apparatus that has a configuration in which laser light from a light source is divided into two by a beam splitter, and the divided laser light is guided to a microscope optical system by separate optical fibers, respectively. It is known (for example, refer to Patent Document 1).

JP 2003-270538 A

  When performing fluorescence observation using laser light as excitation light, it is preferable to minimize the intensity of the laser light in order to prevent fading of fluorescence. On the other hand, when applying a light stimulus to a specimen with laser light, high intensity is required for the laser light. When the same laser beam is used for such two applications, an acoustooptic device such as an acoustooptic variable filter (AOTF) that modulates the intensity of the laser beam is used. That is, the output intensity of the light source is set to an intensity suitable for light stimulation, and when the laser light is used for fluorescence excitation, the intensity of the laser light is attenuated by the acoustooptic device.

However, when the attenuation width of the intensity by the acoustooptic element is increased, the resolution of the intensity of the laser light adjusted by the acoustooptic element is lowered. As a result, there is a problem that the intensity of the laser beam for fluorescence observation cannot be finely adjusted.
A method of switching the output intensity of the light source between light stimulation and fluorescence observation is also conceivable, but in this case, the laser light output condition, for example, pointing, emission angle, wavelength, etc. fluctuate, There is a problem in that the coupling with the optical fiber that guides the laser light to the microscope optical system is shifted and observation cannot be performed with the intended laser power.

  The present invention has been made in view of the above-described circumstances, and enables fine adjustment of the intensity of the laser beam and stable observation even when the laser beam is used at a reduced intensity. An object of the present invention is to provide a microscope apparatus that can be used.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a light source unit that outputs laser light, an observation optical system that observes the sample by irradiating the sample with laser light output from the light source unit, and the observation optical system that applies the laser light from the light source unit A microscope apparatus is provided that includes a fiber optical system that guides the light to the optical system, and two modulation units that are arranged in series in an optical path from the light source unit to the observation optical system and modulate the intensity of the laser light.

According to the present invention, the laser light output from the light source unit is guided to the observation optical system by the fiber optical system, so that the specimen can be observed using the laser light by the observation optical system.
In this case, since the intensity of the laser light is modulated in two stages by the two modulation means while being guided from the light source unit to the observation optical system, the intensity of the laser light is greatly attenuated when used. Also, the intensity of the laser beam can be finely adjusted with a fine width. In addition, since it is not necessary to change the output intensity of the laser light from the light source unit, stable observation can be performed while maintaining good coupling between the laser light and the fiber optical system.

In the above invention, at least one of the modulation means may include an acousto-optic element.
By providing at least one acousto-optic element having such a high modulation gradation number, the intensity of the laser beam can be modulated with high resolution.

Moreover, in the said invention, the said acousto-optic element may be arrange | positioned in the front | former stage of the said fiber optical system.
By doing so, wavefront distortion generated in the laser light when the laser light is modulated by the acousto-optic device is removed by the action of the spatial filter of the fiber optical system while being guided through the fiber optical system. Is done. Thereby, the laser beam whose wavefront is adjusted can be guided to the observation optical system.

In the invention described above, one of the modulation means may include an attenuation filter.
By doing in this way, an apparatus structure can be simplified.

Moreover, in the said invention, the said attenuation | damping filter may be arrange | positioned in the back | latter stage of the said fiber optical system.
When the attenuation filter is mechanically moved in order to change the intensity of the laser light modulated by the attenuation filter, the position of the optical axis is likely to shift in the subsequent stage of the attenuation filter. Therefore, when the attenuation filter is arranged in front of the fiber optical system, the coupling efficiency between the laser light and the fiber optical system can be changed by the mechanical movement of the attenuation filter. Therefore, the coupling efficiency can be stabilized by disposing the attenuation filter in the subsequent stage of the fiber optical system.

  According to the present invention, it is possible to finely adjust the intensity of the laser beam and to perform stable observation even when the laser beam is used at a reduced intensity.

1 is an overall configuration diagram of a microscope apparatus according to an embodiment of the present invention. It is a figure which shows the modification of the microscope apparatus of FIG. It is a figure which shows another modification of the microscope apparatus of FIG. It is a figure which shows another modification of the microscope apparatus of FIG.

Hereinafter, a microscope apparatus 1 according to an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the microscope apparatus 1 according to the present embodiment irradiates a light source unit 2 including a plurality of laser light sources 2 a, 2 b,. An observation optical system 3 to be observed, a fiber optical system 4 for guiding laser light from the light source unit 2 to the observation optical system 3, and a first modulation disposed between the light source unit 2 and the fiber optical system 4 Means 5, second modulation means 6 disposed between the fiber optical system 4 and the observation optical system 3, and a first control unit 7 and a second control unit 8 that control the modulation means 5 and 6. And a user interface 9.

  The light source unit 2 includes a mirror 10a that combines a plurality of laser beams output from a plurality of laser light sources 2a, 2b,..., 2n into the same optical path, and a plurality of dichroic mirrors 10b, 10c,. Each of the laser light sources 2a, 2b, ..., 2n outputs laser beams having different wavelengths. The light source unit 2 outputs laser light combined into a single optical path by the mirror 10a and the dichroic mirrors 10b, 10c,.

The observation optical system 3 includes, for example, an optical microscope device, and irradiates the sample with laser light input from the light source unit 2 via the first modulation unit 5, the fiber optical system 4, and the second modulation unit 6. The reflected light or fluorescence from the specimen is received to obtain an image of the specimen.
The fiber optical system 4 includes an optical fiber 11 whose input end is set on the output optical path of the first modulation means 5 and whose output end is set on the input optical path of the second modulation means 6.

  The first modulation means 5 selectively selects and outputs the wavelength included in the laser light input from the light source unit 2 and can adjust the intensity of the output laser light (hereinafter referred to as the first OTF). Also referred to as AOTF5). The wavelength and intensity of the laser light modulated by the first AOTF 5 are controlled by the frequency and amplitude of the high-frequency voltage applied from the first control unit 7.

  The second modulation means 6 is composed of an AOTF (hereinafter also referred to as a second AOTF 6) that adjusts the intensity of the laser beam input from the fiber optical system 4. The intensity of the laser light modulated by the second AOTF 6 is controlled by the amplitude of the high-frequency voltage applied from the second control unit 8.

  The user interface 9 is configured such that the user can set a time sequence in which the wavelength selected by the first AOTF 5 and the timing of switching the wavelength are set. The user interface 9 is configured such that the user can set the intensity of the laser beam for each wavelength selected by the first AOTF 5. The user interface 9 sends conditions set by the user to the first control unit 7 or the second control unit 8. The first control unit 7 and the second control unit 8 apply a high-frequency voltage having a frequency and an amplitude based on conditions set in the user interface 9 to the first AOTF 5 or the second AOTF 6.

Next, the operation of the microscope apparatus 1 configured as described above will be described.
In the microscope apparatus 1 according to the present embodiment, in the light source unit 2, the laser beams output from the plurality of laser light sources 2a, 2b,. Combine and output. Laser light including a plurality of wavelengths output from the light source unit 2 is input to the first AOTF 5. In the first AOTF 5, an arbitrary wavelength set by the user on the user interface 9 is selected as the laser beam, and the intensity of the laser beam having the selected wavelength is attenuated and output to the fiber optical system 4. The laser light guided and output by the fiber optical system 4 is further attenuated by the second AOTF 6 and then input to the observation optical system 3.

  As described above, the laser light input from the light source unit 2 to the observation optical system 3 is switched according to the time sequence set in the user interface 9 by the user, so that the observation optical system 3 can detect a plurality of types of specimens. Get an image. For example, the observation optical system 3 acquires a plurality of types of fluorescent images having different excitation wavelengths.

  Thus, according to the present embodiment, the intensity of the laser light input to the observation optical system 3 is attenuated in two stages by the first AOTF 5 and the second AOTF 6. Thus, even when laser light is input to the observation optical system 3 with low intensity, the intensity of the laser light from each of the laser light sources 2a, 2b,. There is an advantage that the intensity of laser light as excitation light input to the observation optical system 3 can be adjusted with a fine width.

  Further, since the output intensity of the laser light from the laser light sources 2a, 2b,..., 2n can be made constant, the output of the laser light pointing, emission angle or wavelength from the laser light sources 2a, 2b,. It is possible to stabilize the laser light input to the observation optical system 3 while keeping the conditions constant, thereby maintaining good coupling between the laser light and the optical fiber 11. Further, by using AOTF having a large modulation gradation number of the laser light intensity for both the first modulation means 5 and the second modulation means 6, the intensity of the laser light can be modulated with higher resolution. .

  In the present embodiment, both the first modulation means 5 and the second modulation means 6 are made of AOTF. Alternatively, one of the modulation means may be made of an ND filter. . 2 and 3 show microscope apparatuses 100 and 200 according to modifications including ND filters 6a, 6b, and 6c as second modulation means.

  FIG. 2 shows that a second modulation means moves a single ND filter 6a and a movement mechanism (not shown) that moves the ND filter 6a between a position on the optical axis and a position retracted from the optical axis. The structure provided with is shown. In FIG. 3, the second modulation means arranges the ND filters 6a, 6b, 6c and one of the plurality of ND filters 6a, 6b, 6c on the optical axis. , 6c is shown. The structure provided with the movement mechanism which is not illustrated is shown. As the ND filters 6a, 6b, and 6c shown in FIGS. 2 and 3, ND filters whose transmittance varies continuously may be employed.

  As described above, by using the ND filters 6a, 6b, and 6c as one of the modulation means, the configuration can be simplified as compared with the case where the acoustooptic device is used. In addition, since the ND filters 6a, 6b, and 6c are switched to and from the optical path by a mechanical operation, the optical axis is likely to be displaced in the subsequent stage of the ND filters 6a, 6b, and 6c. Further, the optical axis shift also occurs due to the wedges (error of filter parallelism) of the ND filters 6a, 6b, 6c. Therefore, when the ND filters 6a, 6b, and 6c are arranged in the front stage of the fiber optical system 4, a moving mechanism for the ND filters 6a, 6b, and 6c is used to ensure sufficient coupling efficiency with the optical fiber 11. Both extremely high positional accuracy and holding down the wedges of the ND filters 6a, 6b and 6c (increasing parallelism) are required. Therefore, by arranging the ND filters 6a, 6b, and 6c in the subsequent stage of the fiber optical system 4 as shown in FIG. 2 and FIG. 3, it is not necessary to consider the coupling efficiency with the optical fiber 11, so the above requirement Can be relaxed and the configuration can be simplified.

  Further, when AOTF is used as the modulation means 5, the distortion of the laser light generated on the light source side with respect to the fiber optical system 4 is guided through the optical fiber 11 by the optical fiber 11 acting as a spatial filter. Removed in between. Therefore, there is an advantage that the laser beam can be guided to the observation optical system 3 without deteriorating the optical performance.

  Further, in the present embodiment, as shown in FIG. 4, a light stimulation optical system 12 connected in parallel to the observation optical system 3 may be provided for the light source unit 2 ′. The microscope apparatus 300 according to the modification shown in FIG. 4 includes a beam splitter 13 in which the light source unit 2 ′ divides the laser light combined into a single optical path into two. One of the laser beams divided by the beam splitter 13 is guided to the observation optical system 3 and the other is guided to the light stimulation optical system 12. Reference numeral 14 in the figure denotes a mirror that deflects laser light. Between the mirror 14 and the optical stimulation optical system 12, third modulation means 15 (AOTF in the illustrated example) that modulates the frequency and intensity of the other laser light, and optical stimulation from the third modulation means 15. A fiber optical system 16 that guides laser light to the optical system 12 is provided.

  The microscope apparatus 300 configured as described above can simultaneously perform light stimulation and fluorescence observation of a specimen by the light stimulation optical system 12 and the observation optical system 3. Here, in order to efficiently stimulate the specimen, the intensity of the laser light guided from the light source unit 2 ′ to the photostimulation optical system 12 is preferably high. On the other hand, in order to acquire a fluorescence image of the specimen, it is preferable that the intensity of the laser beam guided from the light source unit 2 ′ to the photostimulation optical system 12 is low in order to prevent the fading of fluorescence. According to the present embodiment, even if the intensity of the laser beam output from the light source unit 2 ′ is set to a sufficiently high intensity suitable for optical stimulation, the first modulation arranged in series in the front stage of the observation optical system 3 The means 5 and the second modulation means 6 can adjust the intensity at a sufficiently high resolution while attenuating the intensity of the laser light input to the observation optical system 3 sufficiently low.

In the present embodiment, one of the two modulation means 5 and 6 is arranged in the front stage of the fiber optical system 4 and the other is arranged in the rear stage of the fiber optical system 4. Both of them may be arranged downstream of the fiber optical system 4.
By doing in this way, when connecting a light source device and a microscope apparatus by the fiber optical system 4 and constructing a microscope apparatus, a light source device can be reduced in size. That is, the light source device is composed of the light source unit 2, and the microscope device is composed of the observation optical system 3 and the two modulation means 5 and 6.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Light source part 2a, 2b, ..., 2n Laser light source 3 Observation optical system 4 Fiber optical system 5 1st modulation means, AOTF (acoustic optical element)
6 Second modulation means, AOTF (acousto-optic element)
6a, 6b, 6c ND filter (modulation means)
7 First controller 8 Second controller 9 User interface 10a Mirror 10b,..., 10n Dichroic mirror 11 Optical fiber 12 Optical stimulation optical system 13 Beam splitter 14 Mirror 15 Third modulation means 16 Fiber optical system

Claims (5)

A light source unit for outputting laser light;
An observation optical system for observing the specimen by irradiating the specimen with laser light output from the light source unit;
A fiber optical system for guiding the laser light from the light source unit to the observation optical system;
A microscope apparatus comprising two modulation means that are arranged in series in an optical path from the light source unit to the observation optical system and modulate the intensity of the laser light.   The microscope apparatus according to claim 1, wherein at least one of the modulation units includes an acousto-optic element.   The microscope apparatus according to claim 2, wherein the acousto-optic element is disposed in front of the fiber optical system.   4. The microscope apparatus according to claim 2, wherein the other modulation means includes an attenuation filter.   The microscope apparatus according to claim 4, wherein the attenuation filter is disposed at a subsequent stage of the fiber optical system.

Images