JPH0476512A - Image pickup method in scanning type microscope - Google Patents

Abstract

PURPOSE:To prevent an image from blurring by detecting illumination light passing specific positions inside assumed innermost main scanning ends and starting sampling operation on every scan once the passage is detected. CONSTITUTION:Two reflected patterns 61a and 61b at the positions B1 and B2 inside the assumed innermost scanning ends A1' and A2' of laser light 58 are slit and the parts are made low in reflection factor, so that the output S4 of a photodetector 62 varies periodically. This signal S4 is inputted to a sampling clock generating circuit 35 to generate a sampling clock S3, which is inputted to an A/D converter 51 to prescribe the sampling period of an analog image signal. A wave-shaped signal S9 is generated with the signal S4 and one pulse rises every time the positions B1 and B2 are passed. Then the image signal begins to be sampled when the pulse is inputted, and consequently even if a scanning end varies, the image signal begins to be sampled without fail when the positions B1 and B2 are passed, so the image having no blur is obtained.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a method for taking a microscopic image of a sample using an optical scanning microscope, and more particularly, it relates to a method for taking a microscopic image of a sample using an optical scanning microscope. The present invention relates to an imaging method that can prevent distortion of output images.

(Prior Art) Conventionally, illumination light is focused on a minute light spot, and this light spot is scanned two-dimensionally on a sample, and at that time, the light that passes through the sample, or the light that is reflected thereon, and Optical scanning microscopes are known in which fluorescence generated from a sample is detected by a photodetector to obtain an electrical signal carrying an enlarged image of the sample. For example, Japanese Patent Laid-Open No. 82-217218 discloses an example of this scanning microscope.

In conventional optical scanning microscopes, a mechanism in which the illumination light beam is two-dimensionally deflected by an optical deflector is often used as the illumination light scanning mechanism.

In addition, the illumination light beam should not be deflected, but should be directed toward the sample stage by 2
It has also been considered to scan the illumination light spot by moving it dimensionally. Furthermore, the applicant's patent application No. 1-2
As shown in the specification of No. 46946, the light transmitting optical system and the light receiving optical system are mounted on a common moving stage, and by moving this moving stage with respect to the sample stage, the illumination light spot is scanned. It is also being considered.

(Problems to be Solved by the Invention) When scanning the illumination light spot by moving the optical system and the sample stage relatively as described above, it is necessary to move the optical system or the sample It is required to move the table at high speed. Therefore, it is conceivable to use, for example, a piezo element, an ultrasonic vibrator, or the like as a drive source for this movement.

However, when moving the optical system or sample stage at high speed in this way, if the drive source such as the piezo element is subjected to disturbances such as temperature, the main scanning trajectory of the illumination light spot will change as shown in Figure 7. However, there are problems in that the main scanning end positions A1 and A2 tend to fluctuate, and the scanning width tends to fluctuate. In particular, when using the piezo element described above, the relationship between the voltage value applied thereto and the displacement is generally not linear, and therefore the above problem is likely to occur even when no external disturbance is applied.

Further, even when the illumination light beam is deflected by an optical deflector to scan the illumination destination light spot, problems similar to those described above may occur due to wobbling or the like of the optical deflector.

On the other hand, in most optical scanning microscopes, the output of a photodetector is periodically sampled to obtain digital image data. Conventionally, the start timing of this sampling in one main scanning period was set at the point when the illumination light was actually located at the main scanning end A1 or A2 (
In the case of one-way main scanning), or at the time of being located at the main scanning end A1 and the time of being located at A2 (in the case of reciprocating main scanning). However, if the sampling start timing is defined in this way, if the illumination light main scanning end position fluctuates as described above, the microscope image output based on the digital image data obtained by this sampling will be distorted. It becomes a thing. In other words, even if the main scanning edge A1 or A2 varies, the output image is reproduced so that the main scanning edge A or A2 is at a constant position in the main scanning direction.

In particular, when the above-mentioned problem occurs when constructing a stereoscopic image from multiple slice images, the obtained image signal must be subjected to pre-correction processing in order to align each slice image with other images. Therefore, the time required to construct a three-dimensional image becomes significantly longer.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an imaging method for a scanning microscope that can prevent image blurring due to fluctuations in the main scanning of illumination light or fluctuations in scanning width.

(Means for Solving the Problems) As described above, the imaging method of the scanning microscope according to the present invention involves main and sub-scanning the illumination light over the sample, and the light from the part of the sample irradiated with the illumination light. In a scanning microscope, the continuous output of the photodetector is sampled at a predetermined period to obtain digital image data, and the illumination on the sample is detected by a photodetector to obtain an image signal representing a microscopic image of the sample. It is characterized by detecting that the illumination light passes through a predetermined position inside the assumed innermost main scanning end of the light, and starting the above-mentioned sampling for each main scanning at the time this detection is made. That is.

(Function) The main scanning locus of the illumination light spot shown in Fig. 8 is the same as that shown in Fig. 7, but from the assumed innermost main scanning end position A, l, A21. If sampling is started when the illumination light passes through the inner B, B2 positions (for example, in the case of reciprocal scanning), the obtained digital image data will be independent of fluctuations in the main scanning end position.
It always shows the image information of the area between the positions Bl and 82.

Therefore, a microscope image output based on such image data can be accurate without blur.

Note that the above position B which defines the effective main scanning width
S % B2 is the assumed innermost main scanning end position A1
It is safer to set the positions B1 and Bz closer to the innermost side than the assumed innermost main scanning edge in order to ensure a large effective main scanning width. It is advantageous to be closer to the positions A1', Az'.

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

1A and 18 show an example of a sampling clock generation device used to carry out the method of the present invention, and FIG. 2 shows a transmission confocal scanning microscope equipped with this device. It shows. First, the basic configuration of the scanning microscope will be explained with reference to FIG. 2 and FIG. 3, which shows the scanning mechanism in detail.

As shown in FIG. 2, a laser diode 5 that emits illumination light 11 is integrally held on a movable table 15.

Furthermore, the moving table 15 includes a light transmitting optical system 8 consisting of a collimator lens 1B and an objective lens 17, and an objective lens 1.
9 and a light receiving optical system 21 consisting of a condenser lens 20,
They are fixed with their optical axes aligned with each other.

A sample stage 22, which is separate from the moving stage 15, is arranged between these optical systems 18, 21. A photodetector 9 is fixed to the movable table 15 below the light receiving optical system 21. As this photodetector 9, for example, a photodiode or the like is used. Further, in front of the photodetector 9 (upper side in the figure), a pinhole plate 8 having a pinhole 8a is arranged.

Laser light (illumination light) emitted from laser diode 5
11 is made into parallel light by a collimator lens 16, and then condensed by an objective lens 17 to a sample stage 22.
The light converges into a minute light spot P on the sample 23 placed on the surface (on the surface or inside it).

The light beams of each transmitted light 11' transmitted through the sample 23 are made into parallel light by the objective lens 19 of the light receiving optical system 21, and then condensed by the condenser lens 20 to form a point image Q.

This point image Q is detected by the photodetector 9. This photodetector 9 outputs a signal S indicating the brightness of each part of the sample 23 irradiated with the light spot P.

Note that by detecting the point image Q through the pinhole 8a, light scattered by the halo and the sample 23 can be cut.

Next, regarding the two-dimensional scanning of the light point P of the illumination light 11, the third
This will be explained with reference to the drawings. The movable table 15 is supported by the pedestal 32 so as to be movable in the direction of arrow X.

That is, one end portions of two guide rods 40.40 are fixed to the pedestal 32, and these guide rods 40.40 are loosely fitted into two guide holes 41.41 provided in the movable table 15. A laminated piezo element 33 is interposed between the movable table 15 and the pedestal 32. This laminated piezo element 33 receives driving power from a piezo element drive circuit 34 and causes the movable table 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 10×103Xi (10XXl0−6×2−2/s).

On the other hand, the sample stand 22 is supported by the pedestal 32 so as to be movable in the direction of the arrow Y, which is perpendicular to the direction of the arrow X. That is, one ends of two guide rods 45.45 are fixed to the pedestal 32, and these guide rods 45.45 are loosely fitted into two guide holes 46.46 provided in the sample stage 22. . A laminated piezo element 47 is interposed between the sample stage 22 and the pedestal 32. This laminated piezo element 47 receives drive power from a piezo element drive circuit 48 and moves the sample stage 22 back and forth in the direction of arrow Y at high speed. Thereby, the sample stage 22 is moved relative to the moving stage 15, and the light spot P sub-scans the sample 23 in the Y direction perpendicular to the main scanning direction X.

Note that the time required for this sub-scanning is, for example, 1/20 second,
In that case, if the sub-scan width is 100 μm, the sub-scan speed is 20XIOCI XIO' -0,002 m/s-2
mm/s, which is sufficiently lower than the main scanning speed. With this level of sub-scanning speed, even if the sample stage 22 is moved, the sample 23 can be prevented from flying away.

By scanning the light spot P two-dimensionally over the sample 23 as described above, an analog signal S carrying a two-dimensional image of the sample 23 is obtained. The creation of pixel-divided digital image data from this image signal S will be explained below with reference to FIG.

FIG. 4 shows the electrical circuit of the scanning microscope.

As shown in the figure, a main scanning drive signal S1 and a sub-scanning drive signal S2 are inputted from the control circuit 35 to the piezo element drive circuits 34 and 48, whereby the main scanning and sub-scanning are synchronized, and the laminated piezo elements 33. 47 is driven.

On the other hand, the analog image signal S output from the photodetector 9 is amplified by an image signal amplifier 50 and then input to an A/D converter 51. The A/D converter 51 also receives a sampling clock S3 from a sampling clock generation circuit 52.
is input, and the A/D converter 51 samples the analog image signal S at the frequency of the sampling clock S3, and converts it into digital image data Sd. This digital image data Sd is stored in the digital image memory 53 and then sequentially read out from there. The read image data Sd is sent to an image reproducing device 54 made of, for example, a CRT, and is provided for reproducing a microscopic image of the sample 23.

Next, the generation of the sampling clock S3 and the points that define the sampling start timing will be explained.

As shown in FIG. 2, a light projecting section 55 is fixed to the movable table 15, and a linear encoder 56 facing the light projecting section 55 is fixed to the pedestal 32. As shown in detail in FIG. 1A, the light projecting section 55 includes a laser diode 57.
, a projection lens 59 that focuses the laser beam 58 emitted therefrom into a minute spot of about 1 to 2 μm, and a projection lens 59 arranged between the lens 59 and the laser diode 57.
60. This laser beam 58
The minute spot is irradiated onto the linear encoder 56. On the linear encoder 56, as shown in FIG. 1B, a large number of reflective patterns 61 are arranged in parallel at a constant pitch of, for example, about 2 μm. These reflection patterns 6
I is made of, for example, a film of aluminum, other metal, or a compound, and reflects the irradiated laser beam 58 with a high reflectance. This linear encoder 56 is attached to the pedestal 32 so that its longitudinal direction, that is, the direction in which the reflection patterns 61 are arranged, is parallel to the main scanning direction X.

When the movable table 15 is reciprocated and main scanning of the illumination light spot P is performed, the laser beam 58 is also transmitted to the linear encoder 5.
6 one-dimensionally reciprocatingly scan. As described above, the illumination light scanning end Al5Az may fluctuate as shown in FIG. 7 due to disturbances acting on the laminated piezo element 33 that drives the moving table 15. The scanning locus 56 of the laser beam 58 on the linear encoder is the sample 23 at the illumination light point P.
Since the scanning locus is the same as the above scanning locus, the above-described variation in the scanning edge occurs in the scanning locus of the laser beam 58 in exactly the same way.

Here, the assumed innermost scanning end Ai of the laser beam 58
', position B1 inside A2' % 2 in BZ
The two reflection patterns Bla and 61b have a shape with a slit in the center, and have a lower overall reflectance than the other reflection patterns.

When the movable table 15 is moved for main scanning of the illumination light spot P, the light projecting section 55 moves in the longitudinal direction relative to the linear encoder 56. Therefore, the laser beam 58 emitted from the light projecting section 55 moves above the linear encoder 56 and alternately irradiates the reflection pattern 61 and the low reflectance portion therebetween.

The laser beam 58 reflected at the reflection pattern 61 is
- reflected by the mirror 80 and detected by the photodetector 62; On the other hand, when the laser beam 58 is irradiating the portion between the reflective patterns 61, the amount of reflected light is significantly reduced. Therefore, the output S4 of the photodetector 62 changes periodically as the moving table 15 moves.

The output signal S4 of the photodetector B2 is amplified by an amplifier 63 as shown in FIG. 4, and then input to the sampling clock generation circuit 52 described above. For example, as shown in detail in FIG. 5, the sampling clock generation circuit 52 includes comparators 84 and 65 to which the amplified signal S4 is input, and a NO signal provided on the output line of the comparator 65.
A T circuit 66, an AND circuit 67 to which the output of this NOT circuit 66 and the output of the comparator 64 are input, and this AN
a capacitor 68 connected to the output line of the D circuit 67;
and P L L to which the output of the comparator 65 is input.
(Phase Locked Loop) circuit 69.

(1) to (6) in FIG. 6 show signal waveforms at each point of the circuit shown in FIG. 5 above. The signal S4 shown in FIG. 6(1) changes periodically as described above.

As shown by arrow a in the figure, when the laser beam 5B irradiates the reflective pattern 61a or 61b (see FIG. 1B), the peak value of the signal S4 decreases due to the decrease in the amount of reflected light.

This signal S4 at comparators 64 and 65, respectively.
The reference signals Vsy and Vsc to be compared with are shown in Fig. 6 (1
). Therefore, the output signal S5 of the converter rotor 4, the output signal S6 of the comparator 65, and the signal S7 obtained by passing the signal S6 through the NOT circuit 66 are respectively (2), (3) in FIG.
The result will be as shown in (4).

The output signal S6 of the comparator 65 is output from the PLL circuit 6.
9 is input. The PLL circuit 69 generates a sampling clock S3 having a frequency f that is N times that of the input signal S6. This sampling clock S3 is input to the A/D converter 51 as described above, and the analog image signal S
Specify the sampling period of

On the other hand, the output signal S8 of the AND circuit 67 to which the above signals S5 and S7 are input has a waveform shown in (5) in FIG. ) is removed, and a signal S9 having a waveform shown in (6) of the same figure is obtained. In other words, in this signal S9, one pulse rises each time the laser beam 58 passes through the positions B1 and B2 shown in FIG. 1B.

This signal S9 is input to the A/D converter 51.

The A/D converter 51 starts sampling the image signal S when the pulse of the signal S9 is input in each illumination light main scan. If you do this, sample 2
3, the scanning ends A1 and A2 of the illumination light spot P are the seventh
Even if it fluctuates as shown in the figure, the sampling of the image signal S always ensures that the illumination light spot P is at a constant position in the main scanning direction (in this example, since it is a reciprocating scan, the position is fixed in each main scanning direction). is different). Note that this fixed position is a position 81%B2 when explained with reference to FIG. 8, and these positions correspond to positions B1 and B2 in FIG. 1B.

The digital image data Sd obtained by specifying the sampling start timing as described above will carry the image information between the fixed positions B1 and 82 on the sample 23 in any case. According to this method, it is possible to output an accurate microscope image without blur.

Although not particularly shown in the figure, the main and sub-scanning directions XS
The sample stage 22 can also be moved in two directions of arrows perpendicular to Y (see FIG. 2), that is, in the direction of the optical axis of the optical system 18.21. If the light spot P is two-dimensionally scanned every time the sample stage 22 is moved a predetermined distance in two directions in this manner, only information on the focused plane is detected by the photodetector 9. Therefore, image data Sd obtained from the output S of this photodetector 9
By loading the sample 23 into the frame memory,
Within the range of movement in the direction, it is possible to obtain image data that represents an image in focus on all surfaces.

It is also possible to construct a stereoscopic image from a plurality of slice images that are shifted from each other in the optical axis direction.

In this case, if the sampling start timing of the image signal S is defined as described above, processing for correcting image blur caused by fluctuations in the scanning edge of the illumination light can be performed between images shifted in the optical axis direction. There will be no need.

In the embodiment described above, only illumination light scan edge fluctuations in the main scanning direction are dealt with, but in addition, illumination light scan edge fluctuations in the sub-scanning direction can also be dealt with in the same manner as described above. Of course, it is also possible.

Further, contrary to the above embodiment, the light projecting section 55 may be fixed to the pedestal 32, and the linear encoder 56 may be fixed to the movable table I5. Furthermore, such a light projecting unit,
In addition to being configured by providing a light source such as a laser diode 57, a configuration may be adopted in which a part of the illumination light 11 is branched and used.

Furthermore, instead of the reflective patterns 61a and 61b in which slits are provided to lower the reflectance as shown in FIG. It is also possible to use a reflection pattern formed using the above method.

Further, the present invention is not limited to a scanning microscope in which an optical system and a sample stage are moved relative to each other using a piezo element, but also to a scanning microscope configured to achieve the above-mentioned relative movement using an ultrasonic transducer, a motor, etc. The present invention can also be applied to a scanning microscope configured to scan an illumination light spot by deflecting an illumination light beam using an optical deflector.

(Effects of the Invention) As explained in detail above, in the imaging method of a scanning microscope according to the present invention, light from a sample portion scanned by illumination light is detected by a photodetector, and a continuous When sampling the output to obtain digital image data, it is detected that the illumination light passes through a predetermined position inside the assumed innermost main scanning edge of the illumination light on the sample, and this detection is performed. At this point, sampling starts for each main scan, so the obtained digital image data always shows image information of a fixed area on the sample, regardless of fluctuations in the main scan end position of the illumination light. becomes. Therefore, according to this method, an accurate microscope image without blur can be reproduced without being affected by the fluctuation of the illumination light main scanning end position.

[Brief explanation of drawings]

FIG. 1A is a perspective view showing an example of a sampling clock generation device for implementing the method of the present invention, and FIG. 1B is a
FIG. 2 is a schematic front view of a scanning microscope equipped with the sampling clock generation device; FIG. 3 is a perspective view showing the main portions of the scanning microscope. 4 is a block diagram showing the electric circuit of the scanning microscope, FIG. 5 is a circuit diagram showing in detail the sampling clock generation circuit of the electric circuit of FIG. 4, and FIG. 6 is a block diagram showing the electrical circuit of the scanning microscope. A graph showing the signal waveform at each point in the circuit, FIG. 7 is an explanatory diagram illustrating fluctuations in the illumination light scanning end position, and FIG. 8 shows fluctuations in the illumination light scanning end position and the sampling start point in the present invention. It is an explanatory diagram explaining the relationship between. 15... Moving table I6... Collimator lens 17. 19... Objective lens 18... Light transmitting optical system 20... Condensing lens 21... Light receiving optical system 22... Sample stage 23 ...Sample 32
... Frame 33.47... Laminated piezo element 34.48... Piezo element drive circuit 50, 83... Amplifier 51... A/D converter 52... Sampling clock generation circuit 55...・
Light projecting section 56... linear encoder 58...
... Laser light 59 ... Projection lens 60 ...
・Half mirror 61...Reflection pattern 64.85...
・Comparator 66...NOT circuit B7...AN
D circuit 68...Capacitor 69...PLL
Circuit 5.57...Laser diode 9.82...Photodetector 11...Illumination light 11'...Transmitted light 1A

Claims (1)

[Claims] Illumination light is caused to main and sub-scan on a sample, and a photodetector detects light from a portion of the sample irradiated with this illumination light to generate an image signal representing a microscopic image of the sample. In a scanning microscope to obtain digital image data, the continuous output of the photodetector is sampled at a predetermined period, and a predetermined position of the illumination light on the sample is located inside the assumed innermost main scanning edge. An imaging method for a scanning microscope, comprising: detecting that illumination light passes through; and starting the sampling for each main scan at the time when this detection is made.