JP2003050354A - Scanning laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a scanning laser microscope with which an irradiation range of a laser beam can capture an observation object in follow-up to a change in the observation object. SOLUTION: The scanning laser microscope which acquires images by two- dimensionally scanning the surface of a focal plane with at least two laser beams varying in wavelengths is furnished with optical systems (1, 2, 4, 6 and 7) constituted in such a manner that at least the two laser beams are aligned to the respective different positions and to the scanning direction of the two-dimensional scanning and are focused onto the focal plane, photoelectric conversion means (14) for detecting the signal light by the laser beams arranged at the head with respect to the scanning direction of at least the two laser beams and control means (18) for controlling the intensity of the laser beam exclusive of the laser beam arranged at the top in accordance with the output signal from the photoelectric conversion means (14).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a scanning laser microscope.

[0002]

2. Description of the Related Art A scanning laser microscope optically slices a sample such as a living cell or tissue to obtain a two-dimensional tomographic image, and further obtains a three-dimensional image from a plurality of two-dimensional tomographic images. It is also possible to obtain. Taking advantage of such characteristics, the scanning laser microscope is used in the fields of physiology, pharmacy, cell biology, etc. to stimulate the cells, for example, to stimulate chemical signals such as electric signals, heat, and chemicals. In the fields of morphology and embryology, it is widely used as a device for observing and recording the detailed structure and shape of cells and the state of deformation and migration over time.

A scanning laser microscope for observing a biological sample creates an image by exciting the fluorescent reagent or fluorescent protein introduced into the sample with laser light and measuring the amount of fluorescence generated thereby. In this method, it is possible to observe a plurality of organs / chemical substances in cells by introducing a plurality of fluorescent substances having different wavelengths of generated fluorescence into the sample. Moreover, in order to excite these plural fluorescent substances, excitation laser beams of plural wavelengths are required.

On the other hand, in the sample, a so-called fading phenomenon occurs in which the amount of fluorescence from the fluorescent substance decreases with the irradiation of the excitation light. For this reason, a scanning laser microscope has been proposed with a mechanism and method for blocking unnecessary excitation light and preventing discoloration as much as possible.

For example, when observing a temporal change of a two-dimensional or three-dimensional image, mechanically in the optical path so that the laser beam is not unnecessarily irradiated to the sample within the interval time from the image acquisition to the next image acquisition. There are methods such as inserting a shutter into the optical disc, attenuating the laser light with an optical filter, an acousto-optical filter, or the like, or electrically shutting off the laser light source.

Further, in Japanese Patent Laid-Open No. 2000-35400, while performing two-dimensional scanning, in a two-dimensional plane,
A method of irradiating, blocking, and modulating laser light is disclosed. According to this method, while scanning the two-dimensional plane with the laser beam, the intensity adjustment of the laser beam and the selection of the wavelength are performed at each position according to the set procedure. As a result, only the desired portion within the two-dimensional plane is irradiated with the laser light of the desired wavelength and intensity.

[0007]

However, in the above-mentioned conventional technique, for example, when the sample to be observed is a cell or the like, and it moves, grows, or deforms with time,
The sample may stick out or move out of a region set to be irradiated with laser light. In such a case, there is a problem that the sample cannot be observed.

It is an object of the present invention to provide a scanning laser microscope which can track a change in the observation target and capture the target observation target in the irradiation range of laser light.

[0009]

In order to solve the problems and achieve the object, the scanning laser microscope of the present invention is constructed as follows.

(1) A scanning laser microscope according to the present invention is a scanning laser microscope which two-dimensionally scans at least two laser beams having different wavelengths on a focal plane to obtain an image. An optical system configured such that light is focused on the focal plane by aligning in different positions and in the scanning direction of the two-dimensional scanning, and at least one of the at least two laser beams is first in the scanning direction. A photoelectric conversion unit that detects the signal light of the arranged laser beam, and a control unit that controls the intensity of the laser beam other than the laser beam arranged at the head based on the output signal from the photoelectric conversion unit. ing.

(2) The scanning laser microscope of the present invention is the microscope according to the above (1), and the at least two laser beams are aligned in the main scanning direction or the sub scanning direction.

(3) The scanning laser microscope of the present invention is the microscope described in (1) or (2) above, and the control means scans the laser beam arranged at the head in the scanning direction. It has mask means for blocking the output signal from the photoelectric conversion means based on the position.

As a result of taking the above-mentioned means, the following effects are achieved.

(1) According to the scanning laser microscope of the present invention, the irradiation range of the laser beam can be set while always detecting the position of the observation target, and even when the observation target moves, grows or deforms. By following the change, the irradiation range of the laser light can catch the target observation object.

(2) According to the scanning laser microscope of the present invention, by arranging at least two laser beams having different wavelengths in the sub-scanning direction, the position of the observation object can be detected more accurately and flexibly. .

(3) According to the scanning laser microscope of the present invention, the position detection range of the observation target is limited, and the observation target can be accurately detected even when a plurality of samples are scattered in the focal plane.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

(First Embodiment) FIG. 1 is a view showing the arrangement of the optical system of a scanning laser microscope according to the first embodiment of the present invention. The optical system of FIG. 1 uses a laser light source 1 and a laser light source 2 as light sources. The laser light source 1 emits laser light having a wavelength (400 to 650 nm) in the visible light range, for example, in order to obtain fluorescence from the sample 11 which is a target signal. The laser light source 2 emits laser light having a wavelength in the infrared region (650 nm or more) that does not excite the fluorescent substance (reagent) introduced into the sample 11.

When a plurality of types of fluorescent substances are introduced into the sample 11 and they are excited at the same time for observation, it is necessary to install a plurality of laser light sources with their optical axes aligned. For the sake of simplicity, the following description will be made assuming that only one laser light source is used to obtain fluorescence by introducing one kind of fluorescent substance into the sample 11.

The respective laser beams from the laser light source 1 and the laser light source 2 pass through the light amount adjusting sections 3 and 5 for controlling the adjustment, emission and blocking of the intensity, and the optical axis synthesizing optical systems 4 and 4 and the excitation light. And the fluorescence from the sample are guided to the XY scanner 7 via the dichroic mirror 6. If, for example, an acousto-optic filter capable of simultaneously controlling laser light of a plurality of wavelengths is used as the light quantity adjusting units 3 and 5, only one such light quantity adjusting unit 3 ′ may be arranged at the position shown in FIG.

The laser light passed by the XY scanner 7 passes through the pupil projection lens 8, the imaging lens 9 and the objective lens 10 to form a spot on the focal plane on the sample 11. Figure 2 here
As described above, the optical axis B of the laser light source 2 has a slight angle Δθ with respect to the optical axis A of the laser light source 1. Then, as shown in FIG. 3, on the focal plane, the main scan (X
A spot 21 is formed by the laser light source 2 at a position displaced by Δd in the (axis) direction.

When the laser light from the laser light source 1 is irradiated, the fluorescent light generated from the fluorescent substance in the sample 11 goes backward in the above optical path and is separated from the excitation laser light by the dichroic mirror 6, and the confocal optical system 15, It is guided to the photoelectric conversion element 17 via 16.

On the other hand, the laser light transmitted through the sample 11 is passed through the condenser lens 12 and the wavelength selection filter 13,
It is guided to the photoelectric conversion element 14. Here, since only the laser light from the laser light source 2 is detected by the photoelectric conversion element 14, for example, a bandpass filter or a longpass filter that transmits only infrared light is used as the wavelength selection filter 13.

The signal detected by the photoelectric conversion element 14 indicates the intensity of the laser light from the laser light source 2 which has passed through the sample 11, and this signal is sent to the signal processing circuit 18. In the signal processing circuit 18, based on the signal from the photoelectric conversion element 14,
In order to judge the state of the sample 11 and adjust the light quantity of the laser light source 1 according to the result, the light quantity adjusting unit 3 or 3'is controlled.

Next, the relationship between the signal obtained from the photoelectric conversion element 14 and scanning will be described. The signal from the photoelectric conversion element 14 is the sample 1 for the wavelength of the laser light from the laser light source 2.
It depends on the transmittance of 1. For example, the cells of the sample 11 to be observed have no transparency to the laser light of the laser light source 2 or are strongly scattered and the photoelectric conversion element 1
When the amount of light incident on 4 is extremely reduced, when the laser spot sequentially scanned in the two-dimensional plane reaches the position where cells are present, the output signal 41 from the photoelectric conversion element 14 changes as shown in FIG. To do.

The signal processing circuit 18 detects a change in the output signal 41 indicated by 411 in FIG.
The laser light of the laser light source 1 is irradiated to the sample 11 until the signal 41 next rises to the threshold 42 or more from the time when it becomes 2 or less, and the light quantity adjusting unit 3 (3 ′) is controlled to generate fluorescence. As a method of detecting a change in the output signal 41, a method in which a threshold value 42 is provided in the signal 41 and a position below or above the threshold value is set as a cell detection position, and a threshold value is set in the amount of change in the signal 41 can be considered. . Other detection methods may be used.

Here, the output signal 41 of the photoelectric conversion element 14
From the time when the laser light source 1 actually emits the laser light until the laser light is emitted from the laser light source 1, the processing time associated with the signal transmission, the signal processing procedure, and the control procedure, and the light quantity adjusting unit 3 (3 ′).
A finite time delay Δt, which is the total of the operation times of the above, occurs. In FIG. 4, this time delay Δt is shown in the output signal 43 from the signal processing circuit 18 to the light quantity adjusting unit 3 (3 ′).

On the other hand, as shown in FIG. 3, two spots 2 displaced by Δd [m] in the main scanning (X axis) direction within the focal plane.
When 0 and 21 are scanned at scan speed v [m / s],
Two spots 20 and 21 are at the same point 1 on the sample 11
When passing over 9, the time difference ΔT shown in the following formula occurs.

ΔT = Δd / v [s] Therefore, the above-mentioned angle Δθ is provided so that ΔT> Δt, and a deviation of Δd is provided between the center position of the spot 20 and the center position of the spot 21. In the signal processing circuit 18, the time delay is adjusted so that ΔT = Δt. As a result, the laser light from the laser light source 1 is transferred to the photoelectric conversion element 14
It becomes possible to irradiate the cells in accordance with the position of the cells detected by.

In general, biological cells are colorless and transparent, and a large change in transmittance cannot be expected for a specific wavelength, and it is often difficult to obtain contrast due to a change in transmittance. Therefore, by incorporating a differential interference optical system (not shown) in the optical system of FIG. 1, it is possible to detect the position of a cell to be observed by a signal with good contrast even when the change in transmittance is small. .

The photoelectric conversion element 17 thus obtained
By displaying the signal from the image as an image of a scanning laser microscope similar to the conventional one, a confocal fluorescence image of only target cells can be obtained. In addition, the photoelectric conversion element 14
The signal from can also be imaged in consideration of the inter-spot distance Δd on the sample surface.

According to the first embodiment as described above,
It is possible to irradiate the laser light that causes the fluorescence only to the position where the observation target exists, and it is possible to suppress the laser irradiation to the unnecessary portion. Furthermore, it becomes possible to set the irradiation range of the laser light while always detecting the position of the observation target, and even if the observation target moves, grows, or deforms, the irradiation range of the laser light is aimed at by following the change. You will be able to capture the observation target.

In the first embodiment, an example in which only one photoelectric conversion element 17 for fluorescence detection is used has been described, but like the excitation laser light source, a plurality of photoelectric conversion elements for fluorescence detection are installed. Also, the same effect can be obtained. Further, the position of the cell may be detected by detecting not the transmitted light but the reflected light from the sample, the autofluorescence of the cell itself, or the fluorescence from the fluorescent substance introduced for the purpose of detecting the position. .

(Second Embodiment) In the first embodiment, as shown in FIG. 5, the optical axis B of the laser light source 2 is relative to the optical axis A of the laser light source 1 with respect to the paper surface. Parallel to Δθ
By tilting it, the spot on the focal plane is Δd in the main scanning direction.
They are staggering. In the second embodiment, by tilting the optical axis B in the direction perpendicular to the paper surface by Δθ (not shown),
As shown in FIG. 5, it is possible to shift the spot by Δd in the sub-scanning (Y-axis) direction. When main scanning (X-axis scanning) is performed in this state, the spot 21 of the laser light source 2 is Δd to the scanning line of the spot 20 of the laser light source 1.
Scanning will be performed on the line that is translated.

Here, if Δd is set to be the same as the sub-scanning interval (scanning line interval), the laser light source 1 is detected from the signal of the photoelectric conversion element 14 by the spot 21 of the laser light source 2.
Thus, the transmitted light information (line profile) of the sample 11 on the line to be scanned next can be obtained from the spot 20 of. Based on this line profile, the signal processing unit 18 determines a control method for the light quantity adjusting unit 3 (3 ′) of the laser light source 1.

For example, data processing is performed on the line profile to remove noise due to dust on the surface of the sample, or pattern matching is applied to the line profile to find a portion that matches a specific signal pattern. It is possible to perform processing such as extraction or enlargement / reduction of the irradiation range of the laser light source 1 with respect to the extracted range.

According to the second embodiment as described above,
As compared with the first embodiment, the light quantity adjusting unit 3 (3 ′) can be controlled more finely, and the observation target can be detected more accurately and flexibly.

(Third Embodiment) In the first and second embodiments, the laser light source 1 for detecting all cells and the like existing in the two-dimensional scanning plane and generating fluorescence is controlled. On the other hand, in the third embodiment, the laser light source 1 is controlled by paying attention to only a part of the cells scattered in the two-dimensional scanning plane.

FIG. 6 is a view showing the arrangement of the optical system of a scanning laser microscope according to the third embodiment of the present invention. 6, the same parts as those in FIG. 1 are designated by the same reference numerals.
In FIG. 6, the scanning position information 22 (scanning position of the spot 21 by the laser light source 2) from the XY scanner 7 and the mask information 23 corresponding to the two-dimensional scanning surface are input to the signal processing unit 18. .

The mask information 23 is information indicating the range within the two-dimensional scanning plane where the output from the signal processing unit 18 to the light quantity adjusting unit 3 (3 ') is valid. This mask information 23 is
For example, it shows the range of the observed cells.
It can be arbitrarily set by the observer according to the observation target. When the scanning position indicated by the input scanning position information 22 is outside the effective range of the mask information 23, the signal processing unit 18 shuts off the output signal from the photoelectric conversion element 14. Also, the signal processing unit 1
When the scanning position is within the effective range of the mask information 23, 8 outputs a light amount adjusting output signal 43 based on the output signal 41 from the photoelectric conversion element 14 to the light amount adjusting unit 3 (3 ′).

Thus, for example, when the cells are scattered in the two-dimensional scanning plane, the laser light from the laser light source 1 can be radiated by paying attention to only a part of the cells.

The present invention is not limited to the above-mentioned respective embodiments, and can be carried out by appropriately modifying it within the scope of the invention.

[0043]

Industrial Applicability The object of the present invention is to provide a scanning laser microscope capable of catching a target observation object in the irradiation range of laser light by following the change even when the observation object moves, grows or deforms. .

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of an optical system of a scanning laser microscope according to a first embodiment of the present invention.

FIG. 2 is a diagram showing a partial configuration of an optical system of the scanning laser microscope according to the first embodiment of the present invention.

FIG. 3 is a diagram showing a positional relationship of spots by two laser light sources of the scanning laser microscope according to the first embodiment of the present invention.

FIG. 4 is a diagram showing an output signal from the photoelectric conversion element and an output signal to a light quantity adjusting unit according to the first embodiment of the present invention.

FIG. 5 is a diagram showing a positional relationship of spots by two laser light sources of the scanning laser microscope according to the second embodiment of the present invention.

FIG. 6 is a diagram showing a configuration of an optical system of a scanning laser microscope according to a third embodiment of the present invention.

[Explanation of symbols]

1 ... Laser light source 2 ... Laser light source 3, 3 '... Light intensity adjustment unit 4. Optical axis synthesizing optical system 5: Light intensity adjustment unit 6 ... Dichroic mirror 7 ... XY scanner 8 ... Pupil projection lens 9 ... Imaging lens 10 ... Objective lens 11 ... Sample 12 ... Condenser lens 13 ... Wavelength selection filter 14 ... Photoelectric conversion element 15, 16 ... Confocal optical system 17 ... Photoelectric conversion element 18 ... Signal processing circuit 23 ... Mask information

Claims (3)

[Claims] 1. A scanning laser microscope that two-dimensionally scans at least two laser beams having different wavelengths on a focal plane to obtain an image, wherein the at least two laser beams are at different positions and at the two-dimensional positions. An optical system configured to be aligned in the scanning direction of scanning so as to be focused on the focal plane, and a signal light from the laser light arranged at the head of the at least two laser lights in the scanning direction is detected. And a control unit for controlling the intensity of laser light other than the laser light arranged at the head based on an output signal from the photoelectric conversion unit. 2. The scanning laser microscope according to claim 1, wherein the at least two laser beams are aligned in the main scanning direction or the sub scanning direction. 3. The control means includes mask means for blocking an output signal from the photoelectric conversion means based on a scanning position of a laser beam arranged at the head in the scanning direction. Item 3. A scanning laser microscope according to Item 1 or 2.