JPH03200915A - Confocal scanning type microscope - Google Patents

Abstract

PURPOSE:To eliminate deterioration of an image signal even without apply correction control over the position shifting of a spot image by making the beam profile of illumination light nearly rectangular, and setting a transmission optical system and a photodetection optical system to satisfy specific relation. CONSTITUTION:This microscope is provided with a beam shaping means which shape the illumination light to have a nearly rectangular beam profile having a flat maximum-light-intensity part. Further, the transmssion optical system and photodetection optical system are related so that d+2epsilon <= D, where (d) is the diameter of an extremely small opening, D is the diameter of the flat maximum light-intensity intensity part of the spot image, and (epsilon) is an estimated maximum value of the quantity of the position shifting of the spot image from the optical axis of the optical systems. Consequently, the spot image can correctly be detected and the microscope with extremely high resolution is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a transmission type confocal scanning microscope, in particular,
The present invention relates to a confocal scanning microscope in which even if the imaging position of a light beam transmitted through a sample is misaligned in a direction perpendicular to the optical axis of an optical system, no adverse effects will be caused by the misalignment.

(Prior Art) Conventionally, illumination light is converged into a minute light spot, and this light spot is scanned two-dimensionally on a sample, and at that time, the light that has passed through the sample or the light that has been reflected there is detected by a photodetector. Optical scanning microscopes are known that detect electrical signals that carry an enlarged image of a 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, and a light transmission optical system that images this illumination light as a minute light spot on the sample. a light-receiving optical system that condenses the light flux from the sample and forms it into a point image; It is composed of a photodetector that detects the point image through an aperture, and a scanning mechanism that two-dimensionally scans the light spot on the sample.

In addition, Japanese Patent Application Laid-open No. 62-217218, JP-A No. 62-209
No. 510 discloses a transmission type microscope as an example of this confocal scanning microscope. This transmission type confocal scanning microscope is configured so that a light beam transmitted through a sample is collected by a light receiving optical system, and a point image thereof is detected by a photodetector.

(Problems to be Solved by the Invention) However, in the above-mentioned transmission type confocal scanning microscope, the laser emission angle of the laser light source used as the illumination light source fluctuates, or the shape and thickness of the sample vary. If the angle changes, the imaging position of the light beam that has passed through the sample may shift in a direction away from the optical axis of the optical system. In this case, this point image and the aperture of the aperture means in front of the photodetector will be misaligned, resulting in a decrease in the S/N of the image signal obtained from the photodetector, and the detection of the light flux that has passed through the sample. Problems arise, such as it becoming completely impossible to do so, or the resolution of the obtained microscopic image being reduced.

In order to prevent such problems, conventional methods have been used, for example, as disclosed in the above-mentioned Japanese Patent Laid-Open No. 62-209510, to detect the positional deviation of the point image and to detect the positional deviation based on the detected positional deviation amount. Therefore, it has been considered to correct the optical path of the light flux toward the photodetector.

Although such a correction method is certainly effective in principle,
For example, since the above-mentioned optical path correction is performed by driving a plane-parallel plate, etc., high-speed response is difficult. If the response speed of this control cannot be made sufficiently high, the scanning speed of the illumination light with respect to the sample must be set low.

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.

Furthermore, if a correction control system is provided to perform the above correction,
Naturally, the equipment becomes complicated and expensive.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a transmission type confocal scanning microscope that does not cause problems such as image signal deterioration even without performing correction control for the positional deviation of the point image.

(Means for Solving the Problems) A confocal scanning microscope according to the present invention includes a sample stage as described above, an illumination light source, a light transmitting optical system, a light receiving optical system, a diaphragm means, and a light receiving optical system. In a transmission type confocal scanning microscope equipped with a detector and a two-dimensional scanning mechanism for a light spot, a beam shaping means is provided for making the beam profile of the illumination light into a substantially rectangular shape having a flat maximum light intensity portion. The diameter of the minute aperture of the aperture means is d, the diameter of the flat maximum light intensity portion in the point image is D1, the maximum value of the expected positional deviation of the point image from the optical axis of the optical system is ε When
It is characterized in that the light transmitting optical system and the light receiving optical system are configured to satisfy the following relationship: d+2 ε≦D.

(Function) FIG. 5 shows the relationship between the beam profile in the above-mentioned point image and the minute aperture of the aperture means. In the figure, a indicates a beam profile, a pinhole plate as a diaphragm means is indicated by 8, and its opening (pinhole) is indicated by 8a. In the same figure (1
)-(a) and (1)-(b), respectively, when the beam profile is rectangular and bell-shaped, the imaging position (point image center) of the light beam transmitted through the sample is on the optical axis C. (2) in the one-way diagram shows a normal state that is consistent with
-(a) and (21-(b)) are for the case where the beam profile is rectangular and the case where it is bell-shaped, respectively.
A state in which the point image center is deviated from the optical axis C by the maximum value ε is shown. As is clear from this figure, when the beam profile is rectangular, if d/2+ε≦D/2, that is, d+2ε≦D, unlike the case where the beam profile is bell-shaped,
There is no variation in the amount of light passing through the pinhole 8a due to point image position deviation.

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

FIG. 1 shows a transmission type confocal scanning microscope according to a first embodiment of the present invention, and FIGS. 2 and 3 show
The scanning mechanism used is shown in detail.

As shown in FIG. 1, a laser diode 5 as a point light source that emits illumination light 11 is integrally held on a movable table 15. This moving table 15 includes a light transmission optical system 18 consisting of a collimator lens 1B and an objective lens 17,
A light receiving optical system 21 consisting of an objective lens 19 and a condensing lens 20 is fixed with their optical axes aligned with each other.

Note that a Gaussian mode filter 6 for beam shaping, which will be described later, is arranged between the collimator lens 16 and the objective lens 17, and a half mirror for focus control, which will be described later, is arranged between the objective lens 19 and the condenser lens 20. -7
are arranged.

Further, a sample stage 22, which is separate from the movable stage 15, is arranged between both optical systems 18 and 21. And light receiving optical system 2
1, both optical systems 18, 21 are mounted on the movable table 15.
A pinhole plate 8 is fixed with the optical axes aligned, and a photodetector 9 is fixed further below.

The above illumination light 11 is made into parallel light by a collimator lens i6, and then condensed by an objective lens 17,
An image is formed on the sample 23 placed on the sample stage 22 (on the surface portion or inside thereof) into a minute light spot P. The luminous flux of the transmitted light 11' that has passed through the sample 23 is converted into parallel light by the objective lens 19 of the light receiving optical system 21, and then converted into parallel light by the condenser lens 20.
The light is focused by , and is focused into a point image Q at the position of the pinhole plate 8 . This point image Q is detected by a photodetector 9 through a pinhole 8a. For example, a photodiode or the like is used as the photodetector 9, from which a signal S carrying an enlarged image of the sample 23 is output.

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 10XIO3X100 XIO' X2-2
m/s.

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 . The time required for this sub-scanning is, for example, 1720 seconds, and in that case, if the sub-scanning width is 100 μm, the sub-scanning speed is: 20×100 XIO°6−0.002 m/s=2
mm/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.

As the light spot P scans the sample 23 two-dimensionally as described above, a time-series signal S carrying a two-dimensional image of the sample 23 is obtained from the photodetector 9. This signal S is made into a pixel-divided signal by, for example, integrating it at every predetermined period.

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 89.
0, it is moved in the two directions of arrows perpendicular to the main and sub-scanning directions X1Y (that is, the optical axis directions of the optical systems 18 and 21). If the light spot P is two-dimensionally scanned each time the two-dimensional moving stage 35 is moved a predetermined distance in two directions in this manner, only information on the focused plane is detected by the photodetector 9. Therefore, by capturing the output S of the photodetector 9 into the frame memory, it is possible to obtain a signal that represents an image that is focused on all surfaces within the range in which the sample 23 is moved in two directions. .

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, thereby synchronizing the main and sub-scanning of the light spot P and the movement of the sample stage 22 in the Z direction.

Next, how to deal with the positional shift of the point image Q will be explained. The Gaussian mode filter 6 shapes the beam profile of the illumination light 11 into a substantially rectangular shape having a flat maximum light intensity portion, as shown in FIG. FIG. 4 shows the light intensity distribution of the illumination light 11 in the direction of the arrow X in the portion of the light point P, but this kind of distribution in all beam cross-sectional directions, including the direction of the arrow Y, of course. It has a light intensity distribution.

The diameter of the flat maximum light intensity portion at the above light point P is D
(See FIG. 4), then the diameter of the flat maximum light intensity portion in the point image Q is also D. The light transmitting optical system 18 and the light receiving optical system 21 satisfy the following relationship: d+2ε≦D, where the diameter of the pinhole 8a is assumed to be the maximum value of the amount of positional deviation of the d1 point image from the optical system optical axis. is configured to be Here, in (1)-(a) and (1)-(b) of Fig. 5, the center of the point image Q is aligned with the optical axis C of the optical system for the case where the beam profile is rectangular and the beam profile is bell-shaped. (2)-(a), (2)-(
b) shows a state in which the center of the point image Q is deviated from the optical axis C by the maximum value ε for the case where the beam profile is rectangular and the beam profile is bell-shaped. When the beam profile is rectangular, as above, if d+2ε≦D, that is, d/2+ε≦D/2, even if the point image position shifts by the maximum value ε, unlike when the beam profile is bell-shaped, , the amount of light passing through the pinhole 8a does not fluctuate due to the point image position shift. In other words, the output signal S of the photodetector 9 that detects the point image Q through the pinhole plate 8 does not change even if the position of the point image Q occurs.

Next, a description will be given of how to deal with the deviation of the point image Q in the optical axis direction. As shown in FIG. 1, a part of the transmitted light 1F that has passed through the sample 23 is split by the half mirror 7. This branched transmitted light 11'' is focused in one direction by a cylindrical lens 50 and then detected by a 4-split photodetector 51. On the other hand, the pinhole plate 8 is moved in the optical axis direction by a piezo element 52. This piezo element 52 is driven by a piezo element drive circuit 53.

When the focusing position of the light point P shifts in the optical axis direction, the imaging position of the point image Q also shifts in the same direction, that is, the pinhole plate 8
It deviates in the vertical direction with respect to the In this case, it becomes impossible for the photodetector 9 to correctly detect the point image Q. Therefore, the four outputs of the four-split photodetector 51 are manually inputted to the focus error detection circuit 54.
4 determines the direction and amount of the focal position deviation (focus error) of the light point P by a so-called astigmatism method based on these outputs. A signal E indicating the direction and amount of this focus error is input to the focus control circuit 55.

The control circuit 55 drives the piezo element 52 so as to move the pinhole plate 8 in one direction of the focus error indicated by the signal E by a distance corresponding to the amount of error. Thereby, the position of the pinhole plate 8 in the optical axis direction is controlled so as to follow the imaging position of the point image Q.

Next, a second embodiment of the present invention will be described with reference to FIG. Note that in FIG. 6, elements that are equivalent to those in FIG.

Illumination light 11 consisting of red light, green light, and blue light is emitted from the R''GB laser IO of this device.

The beam diameter of this illumination light 11 is reduced by a beam compressor 12, condensed by a gradient index lens 13, and made to enter a single mode optical fiber 14.

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. This illumination light 11 is made into parallel light by the collimator lens 1B, passes through the Gaussian mode filter 6 which performs the beam shaping function as described above, is focused by the objective lens 17, and is placed on the sample stage 22. An image is formed into a minute light spot P on the sample 23. Transmitted light 11' transmitted through sample 23
The luminous flux is made into parallel light by the objective lens 19 of the light receiving optical system 21, and then condensed by the condensing lens 20,
The light is introduced into the optical fiber 24 from one end of the single mode optical fiber 24. This optical fiber 24
One end of the lens is fixed to the movable table 15, and a gradient index lens 25 is connected to the other end.

The transmitted light 11' propagated within the optical fiber 24 exits from the other end and is converted into parallel light by the gradient index lens 25.

This transmitted light 1F enters the dichroic mirror 26, and only the blue light 11B is reflected there.
B is detected by the first photodetector 27.

The transmitted light 11' transmitted through the dichroic mirror 26 enters another dichroic mirror 28, and the green light 1
Only 1G is reflected there. This green light 11G
It is detected by a photodetector 29. The transmitted light 11' (ie, red light 11R) transmitted through the dichroic mirror 28 is reflected by the mirror 30 and detected by the third photodetector 31. In this way, the photodetector 27
．． From 29.31 onwards are signals SB, SG, and SR carrying the blue, green, and red components of the enlarged image of the sample 23, respectively.
is output.

In this embodiment, since the end face of the optical fiber 24 constitutes a diaphragm member, the light transmitting optical system
8 and a light receiving optical system 21.

Although the control system for correcting the focus error described above is not particularly shown in FIG. 6, it is of course preferable to provide such a correction control system. In that case, in order to correct the focus error, instead of moving the pinhole plate 8 in the embodiment shown in FIG. 1, the optical fiber 24 should be moved in the axial direction so that the end face position of the optical fiber 24 changes. Bye. In addition, in the apparatus shown in FIG. 6 or in the apparatus shown in FIG. 1, the condenser lens 20 may be moved in the optical axis direction for focus error correction.

Various modifications can be made to the embodiments described above. For example, the pulse motor 37, which is a drive source for reciprocating (sub-scanning) the sample stage 22 fixed on the two-dimensional movement stage 35 in the Y direction, may be a DC motor equipped with an encoder. Instead of sub-scanning the light spot P by moving the moving table 15, the sub-scanning of the light spot P may be performed by moving the movable table 15. 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.

Furthermore, the scanning of the light spot P may be performed by moving the sample stage 22 in two-dimensional directions, as has been conventionally done, without using the moving stage 15 as described above, or by illumination. The light beam may be deflected in two-dimensional directions, or the deflection of the illumination light beam and the movement of the sample stage 22 may be combined.

FIG. 7 shows a third embodiment of the invention in which the illumination light beam is deflected. In this third embodiment device, R
Illumination light 11 consisting of red light, green light, and blue light is emitted from the GB laser IO.

After the beam diameter of the illumination light 11 is adjusted by the beam expander 12°, the illumination light 11 passes through the Gaussian mode filter 6, which performs the beam shaping function as described above, and enters the galvanometer mirror 70, where it is placed at a angle approximately perpendicular to the plane of the paper. deflected in the direction

The deflected illumination light 11 is condensed by the objective lens 17 and is imaged onto a minute light spot P on the sample 23 placed on the sample stage 22 (on the surface or inside thereof). The luminous flux of 1v of transmitted light transmitted through the sample 23 is transmitted to the light receiving optical system 21.
The light is made into parallel light by the objective lens 19, and then enters the galvanometer mirror 71. Galvanometer mirror 7
1 is driven in synchronization with the galvanometer mirror 70, and deflects the transmitted light 11° so as to cancel the deflection of the transmitted light 11' by the galvanometer mirror 70. As a result, the transmitted light 11' after being reflected by the galvanometer mirror 71 travels along a constant optical path, and then passes through the condenser lens 2.
0, the point image Q is focused at the position of the pinhole plate 8.
is imaged.

Transmitted light 11 passing through the pinhole 8a of the pinhole plate 8
' is made into parallel light by the collimator lens 72, and the blue light 11B, green light 11G, and red light 11R are respectively condensed by condensing lenses 73, 74, and 75, and then
It is detected by the photodetector 27, the second photodetector 29, and the third photodetector 31.

The illumination light 11 is deflected as described above to scan the sample 23 one-dimensionally (main scan).

At the same time, the sample stage 22 is reciprocated by a two-dimensional movement stage 35 similar to that of the first embodiment in a direction substantially perpendicular to the main scanning direction (horizontal direction in the figure), and the secondary illumination light 11 is A scan is done.

Next, a fourth embodiment of the present invention will be described with reference to FIG. Although the peripheral portion of the photodetector is omitted in FIG. 8, this portion is essentially formed in the same manner as the device shown in FIG. 7.

In this device, the main scanning galvanometer mirror 7
0, the illumination light 11 enters the sub-scanning galvanometer mirror 76, where the galvanometer mirror 7
It is deflected in a direction approximately perpendicular to the direction of deflection due to zero. Thereby, the illumination light 11 incident on the sample 23 on the sample stage 22
is deflected two-dimensionally, and main and sub-scanning of the illumination light 11 is performed while the sample stage 22 remains fixed.

Then, the transmitted light 11' reflected and deflected by the galvanometer mirror 71 is transmitted to another galvanometer mirror 7.
7. The galvanometer mirror 77 is driven in synchronization with the sub-scanning galvanometer mirror 7B, and deflects the transmitted light 1F so as to cancel the deflection of the transmitted light 1r by the galvanometer mirror 7B.

Thereby, the transmitted light 11' after being reflected by the galvanometer mirror 77 travels along a fixed optical path.

Also in the third and fourth embodiments described above,
The light transmitting optical system 18 and the light receiving optical system 21 are connected to the pinhole 8
The diameter of a is configured to satisfy the relationship d+2ε≦D, where ε is the assumed maximum value of the amount of positional deviation of the d1 point image from the optical axis of the optical system.

As described above in detail, various illumination light spot scanning mechanisms can be applied to the present invention. But the first
If the moving stage 15 is moved to scan the light point P as in the embodiment and the second embodiment, there is no need to move the sample stage 22 at high speed, which prevents the sample 23 from flying away. In addition, high-speed scanning is also possible. Furthermore, if the movable table 15 is used, the illumination light beam will not be swayed, making it easier to design the optical system.
Furthermore, since expensive optical deflectors such as galvanometer mirrors and AODs are not required, the device can be formed at low cost.

(Effects of the Invention) As described above in detail, in the confocal scanning microscope of the present invention, the beam profile of the illumination light is made into a substantially rectangular shape with a flat illumination light intensity portion, and the beam profile is When the diameter of the diaphragm aperture is d, the diameter of the flat ignition light intensity portion in the point image is ε, and the expected maximum value of the positional deviation of the point image from the optical axis of the optical system is ε,
Since the configuration is such that the relationship d+2ε≦D is satisfied, even if the above-mentioned positional deviation occurs, it is possible to correctly detect this point image without being affected by it. Therefore, according to this apparatus, it is possible to always obtain a high-resolution microscopic image.

Furthermore, since the confocal scanning microscope of the present invention does not require a control system for correcting the positional deviation of the point image, it can be manufactured at low cost, and the sample scanning speed can be increased due to the response speed of this control. Therefore, high-speed scanning is possible, and shortening of imaging time is realized.

[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 schematic diagram showing the beam profile of illumination light in the confocal scanning microscope, and FIG. FIG. 6 is a schematic front view showing a confocal scanning microscope according to a second embodiment of the present invention, and FIG. 7 is a third embodiment of the present invention. FIG. 8 is a schematic side view showing a confocal scanning microscope according to a fourth embodiment of the present invention. FIG. 8 is a perspective view showing main parts of a confocal scanning microscope according to a fourth embodiment of the present invention. 5... Laser diode 6... Gaussian mode filter 7... Half mirror 8... Pinhole plate 8a...
Pinhole 9.27.29.31... Photodetector IO... RGB laser 11... Lighting, Meikou 1F
...Transmitted light 14.24...Optical fiber 15...Moving table 1B, 72...Collimator lens 17.19...Objective lens 18...Light sending optical system 20.73.74.75 ...Condensing lens 21
... Light receiving optical system 22 ... Sample stage 23 ...
Sample 26.28...Dichroic mirror 3
0... Mirror 32... Frame 33...
Laminated piezo element 34.53... Piezo element drive circuit 35... Two-dimensional movement stage 3B, 39... Motor drive circuit 37.40... Pulse motor 38... Micrometer 41...
Control circuit 50...Cylindrical lens 51...4-split photodetector 52...Piezo element 54...Focus error detection circuit 55...Focus control circuit 70, 71.7G, 77...Galvanometer mirror Fig. 5 (+) - (b) (2) - (b) Fig. 6 Fig.

Claims (1)

[Scope of Claims] A sample stage on which a sample is placed, a light source that emits illumination light, a light transmission optical system that images the illumination light as a minute light spot on the sample, and a light beam transmitted through the sample. a light-receiving optical system that collects the light and forms it into a point image; a diaphragm member that is arranged such that a minute aperture is located at the position where the point image is formed; and a diaphragm member that detects the point image through the minute aperture. In a confocal scanning microscope equipped with a photodetector and a scanning mechanism that two-dimensionally scans the light spot on a sample, the beam profile of the illumination light is shaped into a substantially rectangular shape having a flat maximum light intensity portion. A beam shaping means is provided, and the diameter of the minute aperture is d, the diameter of the flat maximum light intensity portion in the point image is D, and the amount of positional deviation of the point image from the optical axis of the optical system is assumed. A confocal scanning microscope, wherein the light transmitting optical system and the light receiving optical system are configured to satisfy the following relationship: d+2ε≦D, where ε is the maximum value.