JPH0420912A - Confocal scanning microscope - Google Patents

Abstract

PURPOSE:To scan a light spot at a high speed by holding a light transmission optical system and a light reception optical system on a moving base as one body and moving the moving base back and forth to mainly scan the light spot. CONSTITUTION:A sample base 22 on which a sample 23 is placed, a light source 10 which emits linearly polarized illumination light, a polarization plane maintaining optical fiber 14 where illumination light made incident on one end is propagated and emitted from the other end, a light transmission optical system 18 which forms the image of illumination light emitted from the other end as a minute light spot on the sample 23, and a light reception optical system 21 which condenses the luminous flux from the sample 23 to form its spot image are provided. This device consists of photodetectors 27, 29, and 31 which detect this spot image, a moving base 15 on which the light transmission optical system 18 and the light reception optical system 21 are held as one body, a main scanning means which moves back and fort the moving base 15 so that the light spot is mainly scanned in one direction on the sample 23, and a subscanning means which relatively moves the moving base 15 and a sample base 22 in the direction orthogonal to the direction of an arrow X at a speed lower than the main scanning speed to subscan the light spot on the sample 23.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a confocal scanning microscope, and more particularly to a confocal scanning microscope with an improved light spot scanning mechanism.

(Prior Art) Conventionally, illumination light is converged into a minute light spot, and this light spot is scanned two-dimensionally on a sample. At this time, the light that has passed through the sample or the light that has been reflected there, and also the sample Optical scanning microscopes are known in which the fluorescence emitted from the sample is detected by a photodetector to obtain an electrical signal carrying an enlarged image of the sample.

In particular, it is configured to generate illumination light from a light source and image it into a light spot on the sample, and then re-image the light flux from the sample into a point image, which is then detected by a photodetector. A confocal scanning microscope does not require a pinhole on the sample surface, making it easy to implement.

This confocal scanning microscope basically includes a light source that emits illumination light, a sample stage on which a sample is placed, a light transmission optical system that images this illumination light as a minute light spot on the sample, and the above-mentioned. A light-receiving optical system that collects the light flux (transmitted light, reflected light, or fluorescence) from the sample and forms it into a point image, and a photodetector that detects this point image; It is composed of a scanning mechanism that scans the image. In addition, Tokukai Sho [12-2
17218 discloses an example of this confocal scanning microscope.

(Problems to be Solved by the Invention) In conventional confocal scanning microscopes, the above-mentioned scanning mechanism includes: ■ a mechanism that moves the sample stage two-dimensionally, or ■ a mechanism that moves the illumination light beam two-dimensionally using an optical deflector. A deflection mechanism was used.

However, when the mechanism (2) was adopted, there was a problem that the sample would fly away when high-speed scanning was performed. Many biological samples are observed with microscopes, and if high-speed scanning is not possible when observing these biological samples, it will be impossible to capture the subtle movements of the biological samples. In addition, there is a wide demand for capturing images of samples in near real time, not just for biological samples, and if high-speed scanning is not possible, it is natural that such a demand cannot be met. I can't.

On the other hand, the mechanism (■) allows sufficiently high-speed scanning, but in this mechanism, galvanometer mirrors and AOD
The disadvantage is that an expensive optical deflector such as an acousto-optic optical deflector is required. In addition, in this mechanism (2), since the illumination light beam is deflected by an optical deflector, this light beam enters the objective lens of the light transmission optical system at different angles from time to time, and the resulting aberrations are eliminated. It has also been recognized that the correction makes it difficult to design the objective lens. In particular, when an AOD is used, a special correction lens is required in addition to the objective lens because astigmatism occurs in the light beam emitted from the AOD, making the optical system more complicated.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a confocal scanning microscope that is capable of high-speed scanning, yet has a simple configuration and can be manufactured at low cost.

(Means and Effects for Solving the Problems) A confocal scanning microscope according to the present invention includes a sample stage as described above, a light source, a light transmitting optical system, a light receiving optical system, a photodetector, In a confocal scanning microscope equipped with a two-dimensional scanning mechanism for a light spot, a light source that emits linearly polarized illumination light is used as the light source, and the illumination light input from one end is propagated and emitted from the other end. A polarization-maintaining optical fiber is provided, and the other end of this polarization-maintaining optical fiber is held integrally with the light transmission optical system.
a movable table that guides the illumination light emitted from the other end to the light transmitting optical system and integrally holds the light transmitting optical system and the light receiving optical system; main scanning means for reciprocating the sample so as to perform main scanning in one direction over the sample, and moving the moving stage and the sample stage in a direction substantially orthogonal to the main scanning direction at a speed lower than the main scanning speed. The present invention is characterized in that an illumination light scanning mechanism is constituted by a sub-scanning means for sub-scanning the light spot on the sample by relatively moving the illumination light spot.

Note that the above-described sub-scanning means may be one that moves the movable stage, or, on the contrary, may be one that moves the sample stage. Since the sub-scanning speed can be relatively low, it is possible to prevent the sample from flying away even when the sample stage is moved as described above.

In the above configuration, since the illumination light beam is not swayed for light point scanning, the design of the optical system only needs to be done considering the light rays on the optical axis. It becomes very easy.

In the above configuration, an optical fiber is coupled to the light transmission optical system mounted on the moving table, so that the light source can be placed outside the moving table, so it is possible to reduce the weight of the moving table. .

Furthermore, since a polarization preserving optical fiber is used as the optical fiber, fluctuations in the captured microscopic image can be suppressed.

Note that when the confocal scanning microscope with the above configuration is configured as a reflection type, the light transmitting optical system and the light receiving optical system are shared, and the polarization preserving optical fiber is used for both the illumination light and the light reflected from the sample. will propagate in opposite directions.

On the other hand, when the confocal scanning microscope having the above configuration is constructed as a transmission type microscope, it is desirable that the light receiving optical system and the photodetector are also coupled by an optical fiber in the same manner as described above. Moreover, in that case, it is more preferable to use a polarization-maintaining optical fiber, and the fluctuation of the microscope image can be suppressed in the same way as above.

(Example) Hereinafter, the present invention will be described in detail based on an example shown in the drawings.

FIG. 1 shows a transmission/reflection type confocal scanning microscope according to a first embodiment of the present invention, and FIGS. 2 and 3 show details of the scanning mechanism used therein.

As shown in FIG. 1, an RGB laser 10 emits illumination light 11 consisting of red light, green light, and blue light. This linearly polarized illumination light 11 enters the film surface 12a of the polarizing beam splitter 12 in a P-polarized state and is transmitted therethrough. The illumination light 11 that has passed through the polarizing beam splitter 12 passes through a λ/2 plate 9 for adjusting the polarization plane, and is condensed by a gradient index lens 13 and sent to a polarization preserving optical fiber 1.
4.

As shown in the cross-sectional shape of FIG. 4, this polarization-maintaining optical fiber 14 includes a core 14b disposed within a cladding 14a, and stress applying portions 14C11 on both sides of the core 14b.
The so-called PANDA type is used, in which 4C is formed. The linearly polarized illumination light 11 is λ
/2 By appropriately rotating the plate 9, the direction of the polarization plane is aligned with the direction in which the stress applying portions 14c and 14C are lined up, or with the direction perpendicular thereto (in this embodiment, the direction is the latter direction, that is, the direction shown in FIG. 4). arrow U direction), the optical fiber 1
4.

One end of this optical fiber 14 is fixed to a moving table 15, and the illumination light 11 propagated within the optical fiber 14 is emitted from this one end. At this time, one end of the optical fiber 14 emits illumination light 11 in the form of a point light source. Mobile platform 1
5 includes a collimator lens 16 and an objective lens 17.
A light transmitting optical system 18 consisting of a light transmitting optical system 18 and a light receiving optical system 21 consisting of an objective lens 19 and a condensing lens 20 are fixed with their optical axes aligned with each other.

Further, a sample stage 22, which is separate from the movable stage 15, is arranged between both optical systems 18 and 21. Note that a λ/4 plate 50 is provided between the collimator lens 16 and the objective lens 17.
On the other hand, between the objective lens 19 and the condensing lens 20, there is a
Four plates 51 are respectively arranged.

First, a case where this confocal scanning microscope is used as a transmission type microscope will be described. The above-mentioned illumination light 11 is made into parallel light by the collimator lens 16, passed through the λ/4 plate 50 to become circularly polarized light, and then condensed by the objective lens 17. 23 (on the surface or inside thereof) to form a minute light spot P.

The transmitted light beam of 11° that has passed through the sample 23 is made into parallel light by the objective lens 19 of the light receiving optical system 21, and then
The light is linearly polarized by the /4 plate 51, and then the condensing lens 20
The light is focused by the polarization-maintaining optical fiber 24 and is made to enter the optical fiber 24 from one end thereof. One end of the fiber 24 is fixed to the movable table 15, and a gradient index lens 25 is connected to the other end. Transmitted light 11 propagated within the optical fiber 24
゛ is emitted from the other end, and the gradient index lens 25
is considered to be parallel light.

This transmitted light 11° enters the dichroic mirror 26, and only the blue light 11B is reflected there. The blue light 1
1B is detected by the first photodetector 27 through a halo, a pinhole plate 47 for cutting stray light, and external light. The transmitted light 11° transmitted through the dichroic mirror 26 enters another dichroic mirror 28,
Only the green light 11G is reflected there. This green light 1
1G is detected by the second photodetector 29 through the pinhole plate 48. And the above dichroic mirror 2
Transmitted light 11° transmitted through 8 (i.e. red light 11R)
is reflected by the mirror 30 and detected by the third photodetector 31 through the pinhole plate 49. As the photodetector 27.29.31, for example, a photodiode, a photomultiplier tube, etc. are used, and from these, signals SB, carrying the blue component, green component, and red component of the enlarged image of the sample 23, respectively, are used. SG and SR are output.

On the other hand, when this confocal scanning microscope is used as a reflection type, the reflected light 11° reflected by the sample 23 becomes circularly polarized light with the rotation direction opposite, passes through the λ/4 plate 50, and is polarized. The direction of the wavefront is linearly polarized light orthogonal to that of the illumination light 11. This reflected light beam of 11° is condensed by the frimeter lens 6 and is made to enter the polarization maintaining optical fiber 14. At this time, the direction of the polarization plane of the reflected light 11'' is in the direction of arrow V in FIG. be done.

After passing through the λ/2 plate 9, this reflected light 11' enters the film surface 12g of the polarizing beam splitter 12 in the S-polarized state and is reflected there. This reflected light 11' enters the dichroic mirror 56, and only the blue light 11B is reflected there. The blue light 11B is detected by the fourth photodetector 57 through the pinhole plate 52.

The reflected light 11'' transmitted through the dichroic mirror 56 enters another dichroic mirror 58, and the green light 11''
Only G is reflected there. This green light 11G is detected by the fifth detector 59 through the pinhole plate 53. Then, the reflected light 11° (that is, the red light 11R) transmitted through the dichroic mirror 58 is reflected at the mirror 6a, and passes through the pinhole plate 54 to the sixth photodetector 6.
1 is detected. These photodetectors 57.59.
Signals SB, SG, and SR carrying the blue component, green component, and red component of the enlarged image of the sample 23 are output from 61, respectively.

As mentioned above, the λ/4 plate 50 and the polarizing beam splitter■
2, the reflected light 11" does not return to the RGB laser 10 side, and a larger amount of reflected light II" is transmitted to the photodetector 57.59.
．． 81. In addition, the illumination light 1 reflected by the gradient index lens 13, the end face of the optical fiber 14, etc.
1 is also prevented from entering the photodetector 57, 59, 81, and the signal SB with a high S/N.

You will be able to obtain SG and SR.

In addition, photodetector 27.29.31 for detecting transmitted light 11.
and a photodetector 57.59.81 for detecting reflected light 11° are selectively connected to the subsequent stage via a changeover switch depending on whether the sample 23 is a transmission sample or a reflection sample. Just do it like this.

Furthermore, when the above-mentioned scanning microscope is used as a transmission type, the illumination light 11 is emitted from the light transmitting optical system 18 in a circularly polarized state and enters the light receiving optical system 21 as it is. At this time, there is no need to adjust the planes of polarization between both optical systems 18 and 21. Therefore, the two optical systems 18.21 can be assembled separately, and the two optical systems 18.21 can be combined only by aligning the optical axes, and the assembly of the movable table 15 portion can be done easily.

Next, regarding the two-dimensional scanning of the light point P of the illumination light 11, the second
．． This will be explained with reference to FIG. FIGS. 2 and 3 show the peripheral portion of the movable table 15 as viewed from the upper side and the right side of FIG. 1, respectively. This moving table 15 is held on a pedestal 32 via a laminated piezo element 33. The laminated piezo element 33 receives drive power from the piezo element drive circuit 34, and causes the movable stage 15 to reciprocate at high speed in the direction of arrow X. The frequency of this reciprocating movement is, for example, 10 kHz. In that case, if the main scanning width is 100 μm, the main scanning speed is 10XLO3X100 XIO' X2-2m
/s. Note that since the optical fibers 14 and 24 are bendable, they allow vibration of the movable table 15 while transmitting the illumination light 11, reflected light 11'', and transmitted light 11°, respectively.

On the other hand, the sample stage 22 is fixed to a two-dimensional movement stage 35. This two-dimensional movement stage 35 is driven by a pulse motor 37 that receives a drive current from a motor drive circuit 36.
It is reciprocated in the direction of arrow Y via the micrometer 38. As a result, the sample stage 22 is moved relative to the moving stage 15, and the light spot P moves on the sample 23 in the main scanning direction
Sub-scan in the Y direction perpendicular to . Note that the time required for this sub-scanning is, for example, 1/20 second, and in that case, if the sub-scanning width is 100 μm, the sub-scanning speed is: 20×100 xlO'-o, oo2m/512mm
/s, which is sufficiently lower than the main scanning speed. With this level of sub-scanning speed, it is possible to prevent the sample 23 from flying away even if the sample stage 22 is moved.

By scanning the light spot P two-dimensionally over the sample 23 as described above, continuous signals SB, SG, and SR carrying a two-dimensional image of the sample 23 are obtained.

These signals SB, SG, and SR are made into pixel-divided signals by, for example, sampling at predetermined intervals.

Further, in this embodiment, the two-dimensional moving stage 35 is driven by a pulse motor 4 that receives a drive current from a motor drive circuit 39.
0, it is moved in the direction of arrow Z (that is, the optical axis direction of the optical system 18.21) perpendicular to the main and sub-scanning directions X1Y. In this way, if the two-dimensional scanning of the light spot P is performed every time the two-dimensional moving stage 85 is moved a predetermined distance in two directions, only the information on the focused plane will be transmitted to the photodetector 27, 29, 31 or 57, 59. Detected by .61. Therefore, by importing the outputs SB, SG, and SR of these photodetectors 27.29.31 or 57.59.61 into the frame memory, all of the outputs of the sample 23 in the Z direction can be captured. It becomes possible to obtain a signal that represents an image that is focused on the plane of the image.

Note that the piezo element drive circuit 34 and the motor drive circuit 3B
, 39 are input with a synchronization signal from the control circuit 41, whereby the main and sub-scanning of the light spot P and the movement of the sample stage 22 in two directions are synchronized.

As explained above, in this device, the polarization maintaining optical fiber 14.24 is used as the optical fiber for propagating the illumination light 11, the transmitted light 11°, and the reflected light 11''. ” fluctuations can be suppressed. Therefore, the microscope image reproduced based on the outputs SB, SG, and SR obtained by detecting the transmitted light 11' or the reflected light 11° can be of high quality without distortion.

Various modifications can be made to the embodiments described above. For example, instead of a gradient index lens 13.25,
A microscope objective lens or the like may also be used.

Then, the sample stage 2 fixed to the two-dimensional movement stage 35
The pulse motor 87, which is a drive source for reciprocating the sample stage 22 in the Y direction (sub-scanning), may be a DC motor equipped with an encoder. Instead of doing so, the mobile platform 15
The light spot P may be sub-scanned by moving the light spot P. Further, the movement of the moving table 15 can be performed not only by using the laminated piezo element 33 but also by using, for example, a scanning method using the natural vibration of a solid body caused by a voice coil and ultrasonic waves.

Next, a second embodiment of the present invention will be described with reference to FIG. Note that in FIG. 5, elements equivalent to those in FIG. 1 are given the same numbers, and explanations thereof will be omitted unless necessary.

This confocal scanning microscope is a monochrome reflection type. In this embodiment, the light transmitting optical system and the light receiving optical system are completely shared, thereby realizing a compact confocal scanning microscope with a further simplified optical system.

In this embodiment, a laser diode 70 that emits a monochromatic laser beam is used as a light source that emits the illumination light 11. The reflected light 11'' propagated through the polarization-maintaining optical fiber 14 and emitted therefrom is reflected by the polarization beam splitter 12, and is detected by the photodetector 72 through the pinhole plate 71.

Although the mechanism for scanning the illumination light is not shown in detail in FIG. 5, the one used in the apparatus shown in FIG. 1, for example, may be used as the illumination light scanning mechanism.

In this second embodiment, as well, by providing an optical isolator composed of a λ/4 plate 50 and a polarizing beam splitter 12, the S
/N high signal S can now be obtained.

Also in this case, the polarization maintaining optical fiber 1 is used as an optical fiber for propagating the illumination light 11 and the reflected light 11''.
4, the fluctuations of the illumination light 11 and the reflected light 11' are suppressed, making it possible to reproduce a high-quality microscopic image without unevenness.

Note that the confocal scanning microscope of the present invention can of course be configured exclusively for transmission type. In this case, a polarization-maintaining optical fiber is used as the optical fiber for propagating the illumination light, and more preferably, a polarization-maintaining optical fiber is also used for propagating the transmitted light from the sample. It is possible to suppress the fluctuation of light.

(Effects of the Invention) As explained in detail above, in the confocal scanning microscope of the present invention, the light transmitting optical system and the light receiving optical system are integrally held on a movable stage, and this movable stage is moved back and forth. Since the main scanning of the light spot is configured, there is no need to move the sample stage at high speed, so it is possible to prevent the sample from flying away, and high-speed scanning is also possible.

In the confocal scanning microscope of the present invention, since the illumination light beam is not deflected, the optical system can be easily designed, and an expensive optical deflector such as a galvanometer mirror or AOD is not required, resulting in a simple structure. Therefore, this device can be manufactured at a lower cost than conventional confocal scanning microscopes.

Furthermore, in the confocal scanning microscope of the present invention, an optical fiber is coupled to the light transmission optical system mounted on the movable table, so that the light source can be placed outside the movable table, which reduces the weight of the movable table. Is possible. Therefore, it is possible to significantly speed up illumination light scanning, and it is possible to significantly shorten the time required to capture a microscope image.

Furthermore, in this apparatus, since a polarization maintaining optical fiber is used as the optical fiber, fluctuations in the light propagating through the fiber are suppressed, and a high-quality microscope image can be reproduced.

[Brief explanation of drawings]

FIG. 1 is a schematic front view showing a confocal scanning microscope according to a first embodiment of the present invention, and FIGS. 2 and 3 are a plan view and a side view, respectively, showing essential parts of the confocal scanning microscope. , FIG. 4 is a cross-sectional view of a polarization preserving optical fiber used in the above confocal scanning microscope, and FIG. 5 is a schematic front view showing a confocal scanning microscope according to a second embodiment of the present invention. 10...RGB laser 11...Illumination light 11'
...Transmitted light 11" ...Reflected light 11B...
・Blue light 11G...Green light 11R...Red light 14.24...Polarization preserving optical fiber 15...Moving table 16...Collimator lens 17.19...Objective lens 18... Light transmitting optical system 20... Condensing lens 21... Light receiving optical system 22... Sample stage 2
3...Sample 2B, 28.56.58...Dichroic mirror 2
7.29.31.57.59.61.72... Photodetector 30.60... Mirror 32... Frame 33
...Laminated piezo element 34...Piezo element drive circuit 35...Two-dimensional movement stage 36.39...Motor drive circuit 37.40...Pulse motor 38...Micrometer 41...
Control circuit 47.48.49.52.53.54.71・
...Pinhole plate 50.51...λ/4 board 70
・・・Laser diode diagram diagram diagram diagram diagram diagram

Claims (2)

[Claims] (1) A sample stage on which a sample is placed, a light source that emits linearly polarized illumination light, a polarization-maintaining optical fiber that propagates the illumination light input from one end and emits it from the other end, and this polarization-maintaining optical fiber. a light transmission optical system that is held integrally with the other end of the optical fiber and that images the illumination light emitted from the other end as a minute light spot on the sample; and a light transmission optical system that focuses the light flux from the sample into a point image. a light-receiving optical system that forms an image; a photodetector that detects the point image; a movable table that integrally holds the light-transmitting optical system and the light-receiving optical system; a main scanning means for reciprocating the upper part so as to perform main scanning in one direction, and moving the moving stage and the sample stage in a direction substantially perpendicular to the main scanning direction,
a confocal scanning microscope comprising sub-scanning means for sub-scanning the light spot on the sample by relatively moving at a speed lower than the main-scanning speed. (2) The light transmitting optical system and the light receiving optical system are shared,
The configuration is such that the reflected light from the sample enters the polarization-maintaining optical fiber from the other end, and the reflected light emitted from one end of the polarization-maintaining optical fiber is branched from the optical path of the illumination light toward the optical fiber. 2. The confocal scanning microscope according to claim 1, further comprising an optical isolator that allows the confocal scanning microscope to rotate.