JPH04156507A - Common focal point scanning type phase difference microscope - Google Patents

Abstract

PURPOSE:To perform a high-speed scan without using an expensive light deflector by integrally holding a light transmitting optical system, a light receiving optical system and a phase difference optical system on a moving base, and reciprocating the moving base for a main scan of a light spot. CONSTITUTION:A moving base 15 holding a light transmitting optical system 18, a light receiving optical system 21 and a phase difference optical system is held on a frame 32 via a laminated piezoelectric element 33. The element 33 receives the drive power from an element driving circuit 34 and reciprocates the moving base 15 at a high speed in the arrow X direction. If the reciprocating frequency is set to 10kHz and the main scanning width is set to 100mum, the main canning speed becomes 2m/s. A sample bed 22 is fixed to a two-dimensionally moving stage 35, and it is reciprocated by a pulse motor 37 via a micrometer 38 in the arrow Y direction. A light spot P is sub-scanned on a sample 23 in the Y direction perpendicular to the main scanning direction X. If the required time for a sub-scan is set to 1/20sec and the sub-scanning width is set to 100mum, the sub-scanning speed becomes 2mm/s which is sufficiently lower than the main scanning speed, and the sample 23 is prevented from being flown off.

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 phase contrast microscope that visualizes phase information of a transparent sample.

(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 consists of 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 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 consists of a scanning mechanism that scans dimensionally. Furthermore, Japanese Patent Publication No. 1986-21
No. 7218 discloses an example of this confocal scanning microscope.

On the other hand, a phase contrast microscope has been conventionally provided as one type of microscope for observing transparent objects (phase objects). This phase-contrast microscope basically has a phase plate such as a λ/4 plate placed on opposite sides of the sample, and a ring aperture. A phase delay is given to only one side of the reflected non-diffracted illumination light, and the phase is increased by +6, thereby converting the phase information of the sample into brightness and darkness.

(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 widespread demand for capturing images of specimens in almost real time, not only for biological samples, and if high-speed scanning is not possible, it is natural that this 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.

In the past, attempts have also been made to configure the above-mentioned phase contrast microscope as a confocal scanning type, but in that case, the same problems as described above also occur.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a confocal scanning phase contrast 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 phase contrast microscope according to the present invention includes the above-mentioned sample stage, light source, light transmitting optical system, light receiving optical system, and photodetector. and,
In a confocal scanning microscope equipped with a two-dimensional scanning mechanism for a light spot, a λ/4 beam constituting the phase contrast optical system as described above is used.
A phase plate such as a plate and a ring diaphragm are arranged, one on the light transmitting optical system side and the other on the light receiving optical system side, and the light transmitting optical system, the light receiving optical system, and the phase difference optical system are integrated. a movable stage for holding; a main scanning means for reciprocating the movable stage so that the light spot scans the sample in one direction; The scanning mechanism is characterized by comprising a sub-scanning means that sub-scans the light spot on the sample by moving the light spot relatively in an orthogonal direction at a speed lower than the main-scanning speed.

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.

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

FIG. 1 shows a transmission confocal scanning microscope according to a first embodiment of the invention, and FIGS. 2 and 3 show in detail the scanning mechanism used therein. As shown in FIG. 1, a monochromatic laser IO emits a laser beam 11 as illumination light. The beam diameter of this illumination light 11 is adjusted by a beam compressor 12, passed through a λ/2 plate 9 for polarization adjustment, and then passed through a gradient index lens 13.
The light is focused and input into the polarization maintaining 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. 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.

A ring diaphragm 7 is disposed between both lenses 1B and 17 of the light transmitting optical system 18, and both lenses 1 of the power receiving optical system 21
A ring-shaped phase plate (λ/4 plate) 8, which together with the ring diaphragm 7 constitutes a phase difference optical system, is arranged between 9 and 20. Note that the ring diaphragm 7 is arranged on the front focal plane of the objective lens 17, and the phase plate 8 is arranged in a conjugate relationship with the ring diaphragm 7.

The above-mentioned illumination light 11 is made into parallel light by the collimator lens 1B, made into annular light by the ring diaphragm 7, and then condensed by the objective lens 17. The image is formed on a light point P. Sample 23
The transmitted light (non-diffracted light) having an angle of 11° is converted into parallel light by the objective lens 19 of the light receiving optical system 21, and is incident on the phase plate 8. This phase plate 8 is arranged so as to exactly overlap the transmitted light (non-diffracted light) II' which is annular light. In other words, the inner diameter and outer diameter of this phase plate 8 are equal to the inner diameter and outer diameter of the ring diaphragm 7, respectively, and the objective lens 17.
The value is multiplied by a multiplier of 9. And this phase plate 8
A thin layer is formed on the surface portion of the light beam to appropriately absorb the undiffracted light 11'. On the other hand, sample 23 diffracts and the phase becomes λ/
The diffracted light 11'' delayed by about 4 times mainly passes through the outside of the phase plate 8.

The diffracted light 11'' and the undiffracted light 11' are collected by the condenser lens 2.
The light is focused by 0 and interferes with each other to form an image at the position of Q. One end of the polarization-maintaining optical fiber 24 is arranged at this imaging position, and the undiffracted light 11° and the diffracted light 11'' are made to enter the optical fiber 24 from this one end. 1
5, and a gradient index lens 25 is connected to the other end.

The undiffracted light 11' and the diffracted light 11'' propagated within the optical fiber 24 are emitted from the other end and are condensed by the right-hand lens 25 by the above-mentioned refractive index.

These undiffracted light 11' and diffracted light 11' are detected through an aperture pinhole 26 by a photodetector 27 such as a photomultiplier tube. By using the phase difference optical system as described above, the undiffracted light 11' and the diffracted light 11''
The brightness of the image depends on the phase of the diffracted light 11° (that is, according to the phase information of the illumination light irradiated part of the sample 23)
It is subject to change.

Therefore, the photodetector 27 outputs a light amount signal S corresponding to the phase information of the sample 23.

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 I5 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 83 receives drive power from the piezo element drive circuit 34 and causes the moving stage 15 to reciprocate at high speed in the direction of arrow X. The frequency of this reciprocating movement is, for example, 10kHz. In that case, if the main scanning width is 100 μm, the main scanning speed is 1.0X103 XJ, OOXIO''6X2
-2m/s. In addition, since the optical fibers 14 and 24 have flexibility, the illumination light 11 and the scattered light 11" respectively
The vibration of the movable table 15 is allowed while allowing the vibration to propagate.

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 movable stage 15, and the light spot P touches 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, /20 seconds, and in that case, if the sub-scanning width is 100 μm, the sub-scanning speed is 20X 100 X 10' = 0.002 m /
'5-2 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.

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

If an image is reproduced by an image reproduction means such as a CRT based on this signal S, an image showing the phase information of the sample 23 by brightness and darkness can be obtained.

Note that in the apparatus of the above embodiment, the undiffracted light 11'' is passed through the λ/4 plate 8.
It is a positive phase contrast type in which the phase change can be observed brightly because it passes through the phase plate 8. However, by using a phase plate that delays the phase by 3λ/4 (3π/2) instead of this phase plate 8, the phase change can be observed brightly. It is also possible to use a negative phase contrast type in which the change is observed darkly.

Further, in the above embodiment, the undiffracted light 11'' is passed through the phase plate 8, but it is of course possible to pass the diffracted light 11'' through the phase plate.

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 the arrow Z (that is, the optical axis direction of the optical system 18.21) perpendicular to the main and sub-scanning directions XSY. In this way, if the illumination light spot P is two-dimensionally scanned every time the two-dimensional moving stage 35 is moved a predetermined distance in the Z direction,
Only the information of the focal plane is detected by the photodetector 27. Therefore, by capturing the output S of the photodetector 27 into the frame memory, it becomes possible to obtain a signal representing a three-dimensional image 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 36
．． A synchronization signal is inputted to 39 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 the Z direction are synchronized.

Various modifications can be made to the embodiments described above. For example, the gradient index lens 25, which is a condensing element on the side of the light receiving optical system 21, may be a microscope objective lens or the like. The pulse motor 37, which is a drive source for reciprocating (sub-scanning) the sample stage 22 fixed to the two-dimensional movement stage 35 in the Y direction, may be a DC motor equipped with an encoder. Instead of performing sub-scanning of the light spot P by moving the moving stage 1
The light spot P may be sub-scanned by moving the light spot P. Furthermore, in addition to using the laminated piezo element 33 to move the moving table 15, it is also possible to use, for example, a voice coil, a tuning fork, or even a scanning method that uses the natural vibration of a solid body caused by ultrasonic waves.

Next, referring to FIG. 4, a confocal scanning phase contrast microscope according to a second embodiment of the present invention will be described.

Note that in FIG. 4, parts common to those in FIGS. 1 to 3 are given the same numbers, and detailed explanations thereof will be omitted (the same applies hereinafter).

In this embodiment, contrary to the first embodiment, the light transmission optical system 18
A phase plate of 8 degrees is arranged on the side, and a ring aperture of 7 degrees is arranged on the light receiving optical system 21 side. The phase plate 8'' is formed such that an annular portion 8a delays the phase of the illumination light 11 by λ/4 with respect to other portions, and a thin layer that absorbs the illumination light 11 is provided in the portion other than the portion 8a. is formed. On the other hand, the swing diaphragm 7' has an annular part 7a that blocks light, and the transmitted light (non-diffracted light) 11' that has passed through the above-mentioned part 8a and passed through the sample 23 is just incident on the above-mentioned part 7a. It is arranged so that That is, the phase plate 8' is arranged at the front focal plane of the objective lens 17, and the ring aperture 7° is arranged in a conjugate relationship with the phase plate 8'.

In the above configuration, the transmitted light (non-diffracted light) 11', which is transmitted through the sample 23 after its phase is delayed by λ/4 by the portion 8a of the phase plate 8', is blocked by the ring diaphragm 7'. Therefore, the transmitted light (non-diffracted light) 11゛ that passes through the sample 23 after passing through a portion other than the portion 8a of the phase plate 8°, and the sample 23 after passing through the portion 8a and having a phase delay of λ/4.
The diffracted light 11'' that is incident and diffracted is condensed by the condenser lens 20 and interferes with each other.In this case, the diffracted light 11''
Since the phase is delayed by about λ/2 by 1° with respect to the undiffracted light 11', it becomes a negative phase contrast type.

Next, a third embodiment of the present invention will be described with reference to FIG. The confocal scanning phase contrast microscope of this embodiment is of a reflection type, and a beam splitter 6 is disposed between a phase plate 8' and an objective lens 17, similar to that shown in FIG. ing.

Illumination light 1 that has passed through the collimator lens 16 and the phase plate 8'
1 passes through the film surface 6a of the beam splitter 6, enters the objective lens 17, and converges on a light point P.

The reflected light (non-diffracted light) 11R reflected by the sample 28 after its phase is delayed by λ/4 by the portion 8a of the phase plate 8' is reflected at the film surface 6.
After being reflected at a, it is blocked by a ring diaphragm 7.

Therefore, the reflected light (non-diffracted light) IIR that is reflected by the sample 23 after passing through a part other than the part 8a of the phase plate 8'' and the reflected light (non-diffracted light) IIR that passes through the part 8a and is delayed by λ/4 and then enters the sample 23. The diffracted and reflected diffracted light 11'' is reflected by the film surface 6a and then condensed by the condensing lens 20, and interferes with each other.

As explained above, in this device, the objective lens 17 is shared by the light transmitting optical system and the light receiving optical system. As shown in FIG. 5, an aperture pinhole 5 having an opening having a diameter equal to the diameter of the condensed spot by the condensing lens 20 is arranged in front of the light input end of the polarization-maintaining optical fiber 24 on the receiving side. desirable.

Furthermore, as in each of the embodiments described above, the optical fiber 14
．． 24 to the light transmitting optical system 18 and the light receiving optical system 21, respectively, a small device such as a semiconductor laser is used as the light source, and a small device such as a photodiode is used as the photodetector 27 to connect them. It may also be mounted on the moving table 15.

(Effects of the Invention) As explained above in detail, in the confocal scanning phase contrast microscope of the present invention, the light transmitting optical system, the light receiving optical system, and the phase contrast optical system are integrally held on a movable stage. Since the structure is configured so that the moving table is moved back and forth to perform main scanning of the light spot,
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 phase contrast microscope of the present invention,
Since the illumination light beam is not swayed, it is easy to design the optical system, and the structure is simple, eliminating the need for expensive optical deflectors such as galvanometer mirrors and AODs.
This device can be manufactured at a lower cost than conventional confocal scanning phase contrast microscopes.

[Brief explanation of drawings]

FIG. 1 is a schematic front view showing a confocal scanning phase contrast microscope according to a first embodiment of the present invention, and FIGS. 2 and 3 are plane views showing essential parts of the confocal scanning phase contrast microscope, respectively. The figure and side view, and FIGS. 4 and 5 are schematic front views showing confocal scanning phase contrast microscopes according to the second and third embodiments of the present invention, respectively. 6...Beam splitter 7.7°...Ring aperture 8
．． 8'... Phase plate 10... Monochromatic laser 11
...Illumination light 11'...Transmitted light (non-diffracted light) 11'...Diffracted light 1.1 R...Reflected light (non-diffracted light) 14.24...Optical fiber 15
...Moving table 16...Collimator lens 17.1
．． 9... Objective lens 18... Light transmission optical system 2
0... Condensing lens 21... Light receiving optical system 22
...Sample stand 23...Sample 27...
Photodetector 32... Frame 33... Laminated piezo element 34... Piezo element drive circuit 35... Two-dimensional movement stage 3G, 39... Motor drive circuit 37.40... Pulse motor 38...・Micrometer 41...
Control circuit Figure 1 Figure 4

Claims (1)

[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; A light-receiving optical system that condenses light to form a point image, a photodetector that detects this point image, and a phase plate and a diaphragm, one of which is disposed on the light-transmitting optical system side and the other on the light-receiving optical system side. a phase difference optical system that causes only one of the illumination light diffracted by the sample and the non-diffracted illumination light transmitted through or reflected by the sample to interfere with each other after giving a phase delay; and the light transmission optical system and the light reception optical system. a movable table that integrally holds the system and the phase difference optical system; a main scanning means that reciprocates the movable table so that the light spot scans the sample in one direction; and the movable table. the sample stage in a direction substantially perpendicular to the main scanning direction,
A confocal scanning phase contrast 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.