JPH09304700A - Optical scanning type microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope capable of performing signal processing which is not influenced by disturbance, etc., and performing successive processing. SOLUTION: This microscope 1 is provided with a laser light source 2, a scanner unit 30 two-dimensionally scanning a sample 50 with a laser beam, a 2nd scanner unit 20 two-dimensionally scanning every part of a scanning range by the 1st scanner unit 30 all over the scanning range by the scanner unit 30, and a photodetector 11 detecting fluorescence from the sample 50. By providing an arithmetic circuit 40, in such a case, the specified signal processing is performed based on plural signals outputted from the photodetector 11 and corresponding to the range where the 2nd scanner unit 20 scans, and the processed result is outputted as one signal corresponding to the scanning range by the 2nd scanner unit 20.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an optical scanning microscope.

[0002]

2. Description of the Related Art A conventional optical scanning microscope comprises a laser light source, a plurality of optical scanning means for two-dimensionally scanning a laser beam on a sample, and a light receiving element for detecting light from the sample. The detection signal obtained from the light receiving element is detected according to the scanning pattern of the laser light obtained by scanning the laser light from the laser light source with the plurality of light scanning means to irradiate the sample and scanning with the plurality of light scanning means. A technique of performing signal processing on data supplied to a monitor or the like after photoelectrically converting by a light receiving element to obtain an image signal of a sample is known from JP-A-6-300974.

[0003]

In this conventional optical scanning microscope, when a two-dimensional image of a sample is displayed on a monitor, the data supplied to the monitor is temporarily stored in a memory as a digitized image signal. Image signal to the CPU
It is obtained by signal processing by control of (microcomputer) or arithmetic circuit.

Therefore, there is a problem that the sequential processing cannot be performed, the processing speed becomes slow, and the cost becomes high.

Further, since the point light source is used to obtain one data for one pixel on the monitor screen, when the data is disturbed, it cannot be dealt with and the signal processing is performed. Becomes inaccurate.

The present invention has been made in view of the above circumstances, and its object is to perform signal processing without being affected by disturbances, etc., and to provide a high-speed and inexpensive optical scanning microscope. Is to provide.

[0007]

In order to solve the above-mentioned problems, an optical scanning microscope according to a first aspect of the present invention comprises a light source which emits a light beam and a two-dimensional scanning of the light beam on a sample. One scanning means, a second scanning means for two-dimensionally scanning a part of the scanning range of the first scanning means over the entire scanning range of the first scanning means, and light from the sample In a light scanning microscope equipped with a detection means for detecting, a predetermined signal processing is performed based on a plurality of signals output from the detection means and corresponding to a range scanned by the second scanning means, and a processing result is obtained. Is provided as a single signal corresponding to the scanning range.

Since predetermined signal processing is performed based on a plurality of signals even when there is a disturbance in the signal, the signal processing can be performed with little influence of the disturbance and the reliability of the data is high. Further, after storing all the signals output from the detecting means, the processing is not performed, but a part of the data is processed, so that the sequential processing can be performed.

An optical scanning microscope according to a second aspect of the present invention is the optical scanning microscope according to the first aspect, wherein the second scanning means has a two-dimensional array of light emitting portions for sequentially switching light emission. The detection means is characterized in that the light receiving portions are two-dimensionally arranged corresponding to the light emitting portions.

Since the second scanning means electrically scans not mechanically, the scanning can be performed at high speed.

An optical scanning microscope according to a third aspect of the present invention is the optical scanning microscope according to the first or second aspect,
The second scanning means is a laser diode array,
The detection means is a photodiode array.

The second scanning means can perform high-speed and high-precision scanning by sequentially switching the light emission of the laser diode array at high speed.

An optical scanning microscope according to a fourth aspect of the present invention is the optical scanning microscope according to any one of the first to third aspects, in which the objective lens disposed between the scanning means and the sample is The feature is that the pinhole is arranged at a position conjugate with the focal plane.

Since the pinhole is arranged at a position conjugate with the focal plane of the objective lens arranged between the scanning means and the sample, it can be used as a confocal microscope.

[0015]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a block diagram of an optical scanning microscope according to the first embodiment of the present invention.

The optical scanning microscope 1 according to the present invention comprises a laser light source 2, an excitation filter 3, and a beam expander 4.
A dichroic mirror 5, a second scanner unit (second scanning means) 20, a first scanner unit (first scanning means) 30, a condenser lens 6, an objective lens 7, and a barrier filter. 8, a condenser lens 9, an emission filter 11, and a photodetector (detection means)
12 and an arithmetic circuit (detection signal processing unit) 40.

The laser light source 2 emits an excitation laser.

The beam expander 4 expands the laser light to a size that fills the pupil plane of the objective lens 7.

The dichroic mirror 5 does not transmit the excitation laser light but transmits the fluorescence excited by the sample 50.

The second scanner unit 20 comprises a vertical small scanning mirror (eg galvano mirror) 21 and a horizontal small scanning mirror (eg galvano mirror) 22.
A part of the scanning range of the first scanner unit 30 is two-dimensionally scanned over the entire scanning range of the first scanner unit 30.

The first scanner unit 30 comprises a vertical scanning mirror (eg galvano mirror) 31 and a horizontal scanning mirror (eg galvano mirror) 32.

The objective lens 7 focuses the laser light passing through the condenser lens 6 on the sample 50.

The photodetector 12 detects fluorescence,
The detection signal 12a is converted and output.

The arithmetic circuit 40 performs arithmetic processing according to the intended processing based on the detection signal 12a from the photodetector 12.

The operation of the optical scanning microscope having the above configuration will be described.

The excitation laser light emitted from the laser light source 2 is expanded by the beam expander 4 to a size that can fill the pupil plane of the objective lens 7, is reflected by the dichroic mirror 5, and then the vertical small scanning mirror 21, It is guided to the small horizontal scanning mirror 22, the vertical scanning mirror 31, and the horizontal scanning mirror 32, is irradiated onto the surface of the sample 50 through the condenser lens 6 and the objective lens, and is two-dimensionally scanned on the sample 50. At this time, the fluorescent substance in the sample 50 is excited to generate fluorescence.

This fluorescence is converted from the objective lens 7 to the condenser lens 6, the horizontal scanning mirror 32, and the vertical scanning mirror 3.
1, the horizontal small scanning mirror 22 and the vertical small scanning mirror 21 are traversed in the optical path, and are separated from the excitation laser light by the dichroic mirror 5.

The fluorescence passing through the dichroic mirror 5 is received by the photodetector 12 through the barrier filter 8, the condenser lens 9 and the emission filter 11 and converted into the detection signal 12a by the photodetector 12,
The detection signal 12a is output to the arithmetic circuit 40.

FIG. 2 is a diagram for explaining the movement of the laser beam spot of the two-dimensional small scanning, in which the scanning range of the vertical small scanning mirror and the horizontal small scanning mirror is 3 × 3.
An example will be described in the case of a (vertical direction × horizontal direction) spot data measurement range.

Using the vertical small scanning mirror 21 and the horizontal small scanning mirror 22, as shown by a locus bl in FIG.
The laser light spot is two-dimensionally small-scanned in the data measurement range 51 to perform the measurement. During this measurement, the vertical scanning mirror 31 and the horizontal scanning mirror 32 do not operate.

When the two-dimensional small scanning by the vertical small scanning mirror 21 and the horizontal small scanning mirror 22 is completed, the vertical scanning mirror 31 or the horizontal scanning mirror 3 is completed.
2 is used to shift the scanning range of the vertical small-scanning mirror 21 and the horizontal small-scanning mirror 22 (in FIG. 2, the pixel is horizontally shifted by one pixel as shown by the broken line, but it may be vertically shifted. It does not have to be 1 pixel),
Two-dimensional small scanning is performed again by the vertical small scanning mirror 21 and the horizontal small scanning mirror 22.

Similarly, two-dimensional scanning is performed over the entire scanning range of the first scanner unit 30. The scanning is performed by adjusting the scanning frequency of the scanning mirror.

In this case, the resolution is 1 spot to 1 spot.
Although it is the same as when measured in pixels, it is possible to freely adjust the size of the deviation because it is not necessary to match it with the pixel. Further, the scanning range is not limited to the rectangular shape, and any shape can be used.

FIG. 3 is a diagram for explaining an example of a weighting method in the data measurement range.

FIG. 3 shows an example of the emphasis processing for emphasizing the detection data of the laser light spot at the center, which is calculated using the arithmetic constant (FIG.
Then, the central signal (9) and the peripheral signal (-1) are multiplied by the detection signal (density value) 12a from the photodetector 12 at each spot, and the addition circuit 44 adds them to obtain the data 40a. This data 40a is output as the measurement data of the data corresponding spot 52.

Here, the arithmetic constant "-" means a direction (white) where the image is not displayed. Although not shown in the figure, “+” means a display direction (blackened). Also, the numerical value of the operation constant indicates the degree.

Since the arithmetic constants of the data measurement range 51 thus function as a filter, erroneous data caused by disturbances can be relaxed and output, and the reliability of the output data can be improved. it can.

FIG. 4 is a diagram showing an example of the arithmetic circuit.

The arithmetic circuit 40 is composed of a distribution circuit 41, a delay circuit group 42, a weighting circuit group 43, and an addition circuit 44, and can be realized by using, for example, a gate array or CMOS.

The distribution circuit 41 distributes the detection signal 12a from the photodetector 12 to nine (the number of spots in the data measurement range).

The delay time of the delay circuit group 42 corresponds to the scanning frequency of the laser light spot, and the delay time becomes longer as the detection signal inputted first. When the cycle of the scanning frequency is t, the delay circuit 42i has a delay time of 0 and the delay circuit 42h has a delay time of 0.
Is t, the delay time of the delay circuit 42g is 2t, the delay time of the delay circuit 42f is 3t, the delay time of the delay circuit 42e is 4t, the delay time of the delay circuit 42d is 5t, and the delay time of the delay circuit 42c is 6t. The delay time of the delay circuit 42b is 7t, and the delay time of the delay circuit 42a is 8t.

The weighting circuit group 43 includes the delay circuits 42a to 42a.
The weighting circuits 43a to 43i perform predetermined weighting on the output of 42i, for example, as shown in FIG.

The adder circuit 44 adds the weighted detection signals.

The operation of the arithmetic circuit 40 having the above configuration will be described.

The detection signal 12a input from the photodetector 12 is distributed to the delay circuits 42a to 42i in the order input by the distribution circuit 41.

At the same time when the two-dimensional small scanning of the data measurement range is completed, the weighting circuits 43a to 43i weight the detection signals, and the adding circuit 44 performs addition processing. The processing result is output as one data 40a corresponding to the two-dimensional small scan.

The above operation is performed within one cycle of the clock that shifts the detection signal 12a.

By continuously moving the measuring range 51 two-dimensionally in synchronization with the clock, the image after signal processing can be sequentially displayed on the monitor (not shown), so that the morphology of the sample 50 changes. It is possible to observe a change in image or reaction in a clear image (in the case of emphasis processing).

The two-dimensional scanning means is not limited to the galvanometer mirror, but an acousto-optic device (AOD),
A Nipkow disk or a polygon mirror can also be used. The delay circuit group 42 may be a shift register.

FIG. 5 is a block diagram of the optical scanning microscope according to the second embodiment of the present invention. The same parts as those of the optical scanning microscope of FIG.

The optical scanning microscope 10 according to the present invention includes a laser diode array (light source) 60 and an excitation filter 3.
, Beam expander 4, dichroic mirror 5
And a first scanner unit (first scanning means) 30
A condenser lens 6, an objective lens 7, a barrier filter 8, a condenser lens 9, an emission filter 11,
A photodiode array (detection unit) 70 and an arithmetic circuit (detection signal processing unit) 40 are provided.

The laser diode array 60 has laser diodes arranged two-dimensionally. By electrically performing the two-dimensional small scanning of the laser diode, the two-dimensional scanning of the sample 50 can be performed as in the case of mechanically performing the two-dimensional small scanning of the galvanometer mirror.

The photodiode array 70 has photodiodes arranged in the same manner as the laser diode array 60, and receives fluorescence from the two-dimensionally scanned sample 50.

In the second embodiment, an electrically operated laser diode array 60 and a photodiode array 70 are used.
Since and are used, the scanning can be performed at a higher speed than in the case of the first embodiment, and the measurement processing can be speeded up to the speed of the video rate.

In each of the above-mentioned embodiments, the pinhole 13 can be arranged at a position conjugate with the focal plane of the objective lens 7 to be used as a confocal microscope.

The calculation can be performed not only on the change in the two-dimensional plane, but also in the three-dimensional space using the depth of the sample along the time axis or the optical axis of the confocal microscope.

[0058]

As described above, according to the optical scanning microscope of the first aspect of the present invention, since the predetermined signal processing is performed based on a plurality of signals even when there is a disturbance in the signal, the disturbance is caused. It is possible to reduce the influence of, and to perform highly accurate signal processing. Therefore, accurate observation of the sample can be performed. Also, instead of storing all the signals output from the detection means before performing image processing,
Since a plurality of detection signals obtained from a partial region are successively subjected to signal processing, signal processing can be performed at high speed.
Furthermore, since there is no need for means for storing the detection signal from the detection means for detecting the light from the sample, it is possible to provide an optical scanning microscope for image processing at low cost.

According to the optical scanning microscope of the second aspect of the invention, since the scanning is performed electrically rather than mechanically, the scanning can be performed at high speed and the measurement process can be performed quickly.

According to the optical scanning microscope of the third aspect of the present invention, since the light emission of the laser diode array can be sequentially switched at high speed, high-speed and high-precision scanning can be performed, and the measurement process can be performed quickly. be able to.

According to the optical scanning microscope of the invention described in claim 4, since the pinhole is arranged at a position conjugate with the focal plane of the objective lens arranged between the scanning means and the sample, it is used as a confocal microscope. can do.

[Brief description of drawings]

FIG. 1 is a block diagram of an optical scanning microscope according to a first embodiment of the present invention.

FIG. 2 is a diagram illustrating an image processing method.

FIG. 3 is a diagram illustrating an example of a weighting method.

FIG. 4 is a diagram illustrating an example of an arithmetic circuit.

FIG. 5 is a block diagram of an optical scanning microscope according to a second embodiment of the present invention.

[Explanation of symbols]

 1 Optical Scanning Microscope 2 Laser Light Source 11 Photodetector (Detecting Means) 12 Sample 13 Pinhole 20 Second Scanner Unit (Second Scanning Means) 30 First Scanner Unit (First Scanning Means) 40 Arithmetic Circuit (Detection Signal) Processing unit) 60 laser diode array 70 photo diode array

Claims (4)

[Claims] 1. A light source for emitting a light beam, a first scanning means for two-dimensionally scanning the light beam on a sample, and a part of a scanning range of the first scanning means for the first scanning. An optical scanning microscope comprising: a second scanning unit that two-dimensionally scans the entire scanning range of the unit; and a detection unit that detects light from the sample. It is characterized by further comprising a detection signal processing section for performing a predetermined signal processing based on a plurality of signals corresponding to a scanning range of the second scanning means and outputting a processing result as one signal corresponding to the scanning range. Optical scanning microscope. 2. The second scanning means is a two-dimensional array of light emitting sections for sequentially switching light emission, and the detecting section is two-dimensionally arranged for the light receiving sections corresponding to the light emitting sections. The optical scanning microscope according to claim 1, wherein the optical scanning microscopes are arranged. 3. The optical scanning microscope according to claim 1, wherein the second scanning means is a laser diode array, and the detecting means is a photodiode array. 4. The light according to claim 1, wherein a pinhole is arranged at a position conjugate with a focal plane of an objective lens arranged between the scanning means and the sample. Scanning microscope.