JP2010139951A - Optical device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical device obtaining information in a sample with high resolution. <P>SOLUTION: The optical device is provided with: a light source 101 for emitting laser light; a beam splitter 105 being a division means for dividing the light from the light source 101 into signal light and reference light; a wavefront modulation section 106 for modulating the wavefront of the signal light; an objective lens 107 for condensing the wavefront modulated signal light on the sample; a scan mirror 103 for two-dimensionally scanning the distribution of the light condensed on the sample; a detector 109 for detecting the light from the sample; a beam splitter 105 being a composing means for composing the signal light reflected from the sample and the reference light again, to obtain interference light; a pinhole 113 being a removing section for removing scattered light from the interference light; an interference fringe imaging section 115 for obtaining an interference fringe from the interference light; and an analysis section 117 for performing predetermined control with respect to the wavefront modulation section 106 based on the interference fringe. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

   The present invention relates to an optical device, and more particularly to an optical device using a laser as a light source.

  Fluorescence observation using a laser microscope is an important technique for examining the localization of biological tissues and proteins. In this fluorescence observation method, a sample dyed with a fluorescent dye is irradiated in a spot shape with a focused laser beam. While detecting the fluorescence which generate | occur | produced by this, it scans with a laser beam. As a result, information from fluorescence can be acquired two-dimensionally or three-dimensionally. And it is the method of obtaining an observation image by combining fluorescence information.

  When laser light is condensed inside a biological specimen via an optical system, aberration occurs in the collected light depending on a condensing position (depth position). This is because the aberration of the optical system is corrected in a state where the refractive index between the condensing position and the condensing position is reached in advance. In the case of a biological specimen, the refractive index varies depending on the location due to the difference in density distribution and tissue. Also, the observation position (depth direction) is different. For this reason, aberrations occur in the collected light depending on the condensing position.

  Therefore, for example, Patent Document 1 proposes a technique for modulating a wavefront of light by arranging a wavefront modulator in an optical path in order to correct aberration. For example, Patent Document 2 proposes a technique for measuring the amount of wavefront aberration and feeding back the wavefront aberration to the wavefront modulator. Here, the wavefront aberration is caused by the density distribution, tissue distribution, etc. in the specimen to be observed. Further, the amount of wavefront aberration is measured based on the signal light reflected from the inside of the sample.

Japanese Patent Application Laid-Open No. 11-101942 JP 2008-26643 A

  By the way, when condensing a laser beam inside a non-transparent sample, the laser beam not only deteriorates the condensing state when passing through the inside of the sample, but also receives strong scattering. Therefore, the scattered light is included in the signal light returned from the condensing position. In this way, when scattered light is included in the signal light, the sample information is not correctly reflected. However, since the scattered light cannot be removed by the methods of Patent Documents 1 and 2, information inside the specimen cannot be obtained with high resolution.

  The present invention has been made in view of the above, and an object of the present invention is to provide an optical device capable of acquiring information inside a specimen with high resolution.

In order to solve the above-described problems and achieve the object, according to the present invention, a light source that emits laser light,
A splitting means provided at a position where the laser beam is incident, and forming a signal optical path, a reference optical path, and a detection optical path;
An objective optical system disposed on the signal optical path;
A reference member disposed on the reference optical path;
Wavefront modulation means disposed between the dividing means and the objective optical system;
An optical device comprising interference fringe imaging means provided in the detection optical path,
The splitting unit splits the laser light from the light source into signal light traveling toward the objective optical system and reference light toward the reference member, and returns light from the objective optical system and return from the reference member. Combine the light and enter the interference fringe imaging means,
The wavefront modulation means modulates the wavefront of the signal light,
The objective optical system focuses the wavefront modulated signal light on the sample,
Further, a removing unit that is disposed in an optical path from the dividing unit to the interference fringe imaging unit and removes scattered light from the interference light;
Control means for controlling the wavefront modulation means based on the interference fringes;
Can be provided.

  Further, according to a preferred aspect of the present invention, it is desirable to have a scanning unit that is arranged in an optical path from a light source to the dividing unit and that two-dimensionally scans the distribution of light condensed on the sample.

  Further, according to a preferred aspect of the present invention, it is desirable to have scanning means for performing scanning by applying light condensed on the specimen to the entire specimen surface by moving the specimen two-dimensionally.

  Moreover, according to the preferable aspect of this invention, it is desirable that a removal means contains a condensing lens, a pinhole, and a collimating lens.

  Further, according to a preferred aspect of the present invention, it is desirable that the dividing means is composed of one optical element.

  Further, according to a preferred aspect of the present invention, it is desirable that the dividing means is composed of two optical elements.

  Moreover, according to the preferable aspect of this invention, it is desirable that a light source is a pulse laser light source.

  The optical device according to the present invention has an effect that information inside the specimen can be acquired with high resolution.

  Embodiments of an optical device according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

Example 1
FIG. 1 shows a schematic configuration of an optical device 100 according to Example 1 of the present invention. In FIG. 1, the optical device 100 includes a light source 101. The light source 101 generates laser light having a wavelength that excites the fluorescent dye of the specimen 10. A collimating optical system (not shown) is disposed on the optical path along which the laser light travels. The collimating optical system changes the beam diameter of the laser light emitted from the light source 101 into parallel light.

  Light from the light source 101 passes through the ND filter 102 having a light amount modulation function and enters the scan mirror 103. The scan mirror 103 can deflect light in, for example, two orthogonal directions by a drive unit (not shown). Thereby, the laser beam condensed on the specimen 10 (on the focal plane 10a) is scanned in a two-dimensional direction.

  The light reflected by the scan mirror 103 enters the dichroic mirror 104. The dichroic mirror 104 reflects the laser light from the light source 101 in the direction of the sample 10 and transmits light having a predetermined wavelength out of the light from the sample 10, that is, light having a fluorescence wavelength emitted from the sample 10.

  The light reflected by the dichroic mirror 104 in the direction of the sample 10 enters the beam splitter 105. The beam splitter 105 splits the incident light into signal light that transmits in the direction of the specimen 10 and reference light that reflects in the direction of the reference mirror 110. That is, in the first embodiment, the beam splitter 105 corresponds to a dividing unit that divides light from the laser light source into signal light and reference light.

  The signal light traveling in the direction of the specimen 10 will be described. The signal light passes through the transmissive wavefront modulation unit 106. The transmissive wavefront modulation unit 106 can control the light transmittance at an arbitrary position to a predetermined value in a two-dimensional plane perpendicular to the optical axis. Thereby, the phase of the transmitted wavefront can be controlled. As will be described later, the transmission wavefront modulation unit 106 is controlled by a signal from the analysis unit 117. Examples of the transmissive wavefront modulation unit used in the present application include a liquid crystal and a photorefractive crystal.

  The signal light that has passed through the transmissive wavefront modulation unit 106 is collected at the focal position P in the sample 10 by the objective lens 107. Here, by making the tilt of the scan mirror 103 variable, the focal plane 10 a can be scanned with the laser light condensed at the focal position P.

  A part of the signal light is reflected in the condensed minute region at the focal position P of the specimen 10. The reflected signal light again enters the beam splitter 105 after passing through the objective lens 107 and the transmission wavefront modulation unit 106. The signal light reflected by the beam splitter 105 enters the interference fringe imaging unit 115.

  Next, reference light will be described. As described above, the beam splitter 105 splits the incident light into signal light that passes in the direction of the sample 10 and reference light that reflects in the direction of the reference mirror 110. The reference light reflected in the direction of the reference mirror 110 is reflected by the reference mirror 110.

  The reference mirror 110 is placed on the movable stage 111. The light reflected by the reference mirror 110 passes through the beam splitter 105. The signal light reflected by the beam splitter 105 and the reference light transmitted through the beam splitter 105 are combined coaxially. At this time, the movable stage 111 moves the reference mirror 110 in the optical axis direction so that the optical path length of the reference light matches the optical path length of the reflected light.

  In the beam splitter 105, the combined reference light and reflected light cause interference, and enter the interference fringe imaging unit 115. In the interference fringe imaging unit 115, a pinhole 113 is disposed at the focal position of the condenser lens 112. The light condensed by the condenser lens 112 and passing through the pinhole 113 is transmitted through the collimator lens 114 and enters the image sensor 116.

The movable stage 111 is used as an optical path length adjusting unit. The movable stage 111 is moved in a direction along the optical axis. Thereby, the optical path length difference between the signal light and the reference light changes, and the contrast of the interference fringes changes. Then, the position of the reference mirror 110 is adjusted so that an interference fringe having an optimum contrast is obtained from the difference in brightness between the bright part and the dark part. The interference fringe pattern formed by the signal light and the reference light is captured by the image sensor 116.
Note that the movable stage 111 is not necessarily required when the optical path lengths of the reference light and the signal light are adjusted in advance.

  The analysis unit 117 analyzes the interference fringe pattern of the signal light and the reference light. Thereby, the deviation of the wave fronts of the reference light and the signal light is obtained. Then, the analysis unit 117 calculates a wavefront modulation amount that reduces the deviation between the wavefronts. The wavefront modulation amount is sent to the transmissive wavefront modulation unit 106, and the transmissive wavefront modulation unit 106 updates the wavefront modulation according to the calculated wavefront modulation amount.

  The process of the deviation of the wavefronts of the signal light and the reference light, the calculation of the wavefront modulation amount for reducing the deviation, and the display on the transmission wavefront modulation unit 106 are repeated. As a result, the deviation between the wavefronts of the signal light and the reference light is reduced. For this reason, the aberration of the signal light condensed inside the sample 10 is reduced. As a result, it is possible to generate a spot that approaches the diffraction limit inside the sample 10. Therefore, information inside the specimen 10 can be acquired with high accuracy (high resolution) by the imaging element 116.

  Furthermore, since the living tissue is not a completely transparent body, the light propagating through the inside receives very strong scattering. For this reason, the signal light contains a lot of scattered light. These scattered lights are generated at random locations including the area where the generated area is condensing, and the traveling direction is uniformly scattered. Since the surface of the sample and the surface of the pinhole are in a conjugate relationship, even if light is collected by a condenser lens, the scattered light will not be collected and will be scattered over a wider area than the pinhole opening. I can't go through the pinhole. As a result, most of the scattered light is blocked by the pinhole 113. As a result, it is possible to reduce the influence of the scattered light included in the interference fringe pattern of the signal light for measuring the wavefront modulation amount. Therefore, the wavefront aberration can be corrected accurately, and the information inside the sample 10 can be acquired with high resolution.

  Next, of the light reflected from the specimen 10, that is, the signal light that passes through the beam splitter 105 will be described. The signal light transmitted through the beam splitter 105 is light having a fluorescence wavelength. For this reason, it passes through the dichroic mirror 104. The transmitted light is collected on the detector 109 by the condenser lens 108. Thereby, a fluorescence image can be observed.

In the present invention, when the light source 101 is a light source that generates continuous laser light, the specimen 10 can only be observed based on the one-photon absorption principle.
When the light source 101 generates a pulse laser, the specimen 10 can be observed based on the one-photon absorption principle or the two-photon absorption principle.
Observation based on the principle of two-photon absorption is preferable because information up to the deeper part of the specimen 10 can be obtained.

(Example 2)
Next, an optical device 200 according to Embodiment 2 of the present invention will be described with reference to FIG. The same parts as those in the first embodiment are denoted by the same reference numerals, and redundant description is omitted.

  In the present embodiment, the reflection wavefront modulation section 202 is used as a wavefront modulation means instead of the transmission wavefront modulation section 106 of the first embodiment, and the place where the scan mirror 103 is disposed in the first embodiment is fixed. The difference from the first embodiment is that the mirror 103a is changed and the scan mirror 103 is disposed between the reflection type wavefront modulation unit 202 and the objective lens 107. In addition, in order to guide the light from the light source 101 in the direction of the sample 10, in this embodiment, a mirror 201 having a triangular prism with a cross section and two mirror surfaces is used. Note that the mirror 201 may have two mirrors instead of the triangular prism shape. In addition, examples of the reflective wavefront modulation unit used in the present application include a variable mirror and a reflective crystal.

  The analysis unit 117 analyzes the interference fringe pattern of the signal light and the reference light. Thereby, the deviation of the wave fronts of the reference light and the signal light is obtained. Then, the analysis unit 117 calculates a wavefront modulation amount that reduces the deviation between the wavefronts. The wavefront modulation amount is sent to the reflective wavefront modulation element 202.

(Example 3)
Next, an optical device 300 according to Embodiment 3 of the present invention will be described with reference to FIG. The same parts as those in the first embodiment and the second embodiment are denoted by the same reference numerals, and redundant description is omitted.

  In the present embodiment, in place of the beam splitter 105 in the first embodiment, a laser beam splitting unit and a combining unit are separately provided. That is, the optical apparatus 300 is provided with a beam splitter 105a as a dividing unit and a beam splitter 105b as a combining unit. The beam splitter 105a splits the light from the light source 101 into reference light and signal light.

  Of the divided light, the signal light passes through the beam splitter 105 b and enters the sample 10 through the mirror 201, the reflective wavefront modulation unit 202, and the objective lens 107. The signal light having the fluorescence wavelength reflected from the sample 10 is reflected again toward the interference fringe imaging unit 115 by the beam splitter 105b through the objective lens 107, the mirror 201, and the reflection type wavefront modulation unit 202.

  Among the divided lights, the reference light is bent in the optical path by the two mirrors 110a and 110b and enters the beam splitter 105b. The two mirrors 110 a and 110 b are placed on the movable stage 111. The beam splitter 105b has a configuration in which the signal light and the reference light described above are combined and guided to the interference fringe imaging unit 115.

Example 4
Next, an optical device 400 according to Embodiment 4 of the present invention will be described with reference to FIG. The same parts as those in the first embodiment and the second embodiment are denoted by the same reference numerals, and redundant description is omitted.

  In this embodiment, a fixed mirror 103a that bends the optical path of light from the light source 101 is fixed. The specimen 10 is placed on the two-dimensional stage 401. By moving the two-dimensional stage 401 holding the specimen 10, the focal plane 10a of the specimen 10 can be scanned with the laser spot light.

(Example 5)
Next, an optical device 500 according to Embodiment 5 of the present invention will be described with reference to FIG. The same parts as those in the first embodiment and the second embodiment are denoted by the same reference numerals, and redundant description is omitted. In the present embodiment, light is divided and combined using polarized light.

  The light emitted from the light source 101 passes through the (1/2) λ plate 501, the ND filter 102, and the scanning scan mirror 103, and enters the polarization beam splitter 105 </ b> PB that is a dividing unit. The polarization beam splitter 105PB divides incident light into two parts: reflected S-polarized light and transmitted P-polarized light.

  The (1/2) λ plate 501 can change the direction of linearly polarized light emitted from the light source 101. Thereby, the division ratio by the polarization beam splitter 105PB can be changed.

  The S-polarized reference light reflected by the polarization beam splitter 105PB is reflected by the reference mirror 110, which is an optical path length modulation means, and returns to the polarization beam splitter 105PB again. At that time, it passes through the (¼) λ plate 502 twice. For this reason, it is converted from S-polarized light to P-polarized light. As a result, when returning to the polarization beam splitter 105PB, the reference light is transmitted without being reflected.

  On the other hand, the P-polarized signal light that passes through the polarization beam splitter 105PB irradiates the specimen 10 through the mirror 201, the reflective wavefront modulation unit 202, and the objective lens 107. The light returned from the specimen 10 returns to the polarization beam splitter 105PB via the same path again. At that time, similarly to the reference light, the light passes through the (¼) λ plate 503 twice. For this reason, the signal light is converted from P-polarized light to S-polarized light.

  As a result, the signal light is reflected by the polarization beam splitter 105PB and combined with the reference light. Thus, the polarization beam splitter 105PB also functions as a combining unit. The reference light and the signal light synthesized by the polarization beam splitter 105PB are orthogonal in polarization direction. For this reason, no interference occurs as it is. The polarizing plate 504 converts each light into the same polarized light. The wavefront transmitted through the polarizing plate 504 generates interference fringes. Since other parts are the same as those in the first and second embodiments, description thereof will be omitted.

  In this embodiment, polarized light is used. For this reason, the loss of the light quantity by division | segmentation or a composition can be reduced. For this reason, the light from a light source can be used effectively.

  The present invention is useful as an optical apparatus for acquiring information inside a specimen with high resolution after removing the influence of scattered light. The present invention is particularly applicable to a microscope for observing the inside of a living body, an endoscope capable of deep observation, and the like.

It is a figure which shows schematic structure of the optical apparatus which concerns on Example 1 of this invention. It is a figure which shows schematic structure of the optical apparatus which concerns on Example 2 of this invention. It is a figure which shows schematic structure of the optical apparatus which concerns on Example 3 of this invention. It is a figure which shows schematic structure of the optical apparatus which concerns on Example 4 of this invention. It is a figure which shows schematic structure of the optical apparatus which concerns on Example 5 of this invention.

Explanation of symbols

10 Specimen 10a Focal plane P Condensing position 100, 200, 300, 400, 500 Optical device 101 Light source 102 ND filter 103 Scan mirror 103a Fixed mirror 104 Dichroic mirror 105, 105a, 105b Beam splitter 105PB Polarization beam splitter 106 Transmission type wavefront modulation Unit 107 objective lens 108 condensing lens 109 detector 110 reference mirrors 110a and 110b mirror 111 movable stage 112 condensing lens 113 pinhole 114 collimating lens 115 interference fringe imaging unit 116 imaging element 117 analysis unit 201 mirror 202 reflection type wavefront modulation unit 401 Two-dimensional stage 501 (1/2) λ plate 502, 503 (1/4) λ plate 504 Polarizing plate

Claims (7)

A light source that emits laser light;
A splitting means provided at a position where the laser beam is incident, and forming a signal optical path, a reference optical path, and a detection optical path;
An objective optical system disposed on the signal optical path;
A reference member disposed on the reference optical path;
Wavefront modulation means disposed between the dividing means and the objective optical system;
An interference fringe imaging means provided in the detection optical path,
The splitting unit splits the laser light from the light source into signal light traveling toward the objective optical system and reference light toward the reference member, and returns light from the objective optical system and return from the reference member. Combine the light and enter the interference fringe imaging means,
The wavefront modulation means modulates the wavefront of the signal light,
The objective optical system focuses the wavefront modulated signal light on the sample,
Further, a removing unit that is disposed in an optical path from the dividing unit to the interference fringe imaging unit and removes scattered light from the interference light;
Control means for controlling the wavefront modulation means based on the interference fringes;
An optical device comprising:   The optical apparatus according to claim 1, further comprising a scanning unit that is arranged in an optical path from the light source to the dividing unit and that two-dimensionally scans a distribution of light collected on the specimen.   The optical apparatus according to claim 1, further comprising a scanning unit that performs scanning by causing the sample to move in a two-dimensional manner so that light condensed on the sample is applied to the entire surface of the sample.   The optical device according to any one of claims 1 to 3, wherein the removing unit includes a condenser lens, a pinhole, and a collimating lens.   The optical apparatus according to claim 1, wherein the dividing unit includes one optical element.   The optical device according to any one of claims 1 to 4, wherein the dividing unit includes two optical elements.   The optical device according to claim 1, wherein the light source is a pulse laser light source.

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