JPH07139931A - Apparatus for irradiating laser in laser scan microscope - Google Patents

Abstract

PURPOSE:To uniform an amount of a laser light cast to a sample at all positions in a scanning area on a surface of the sample by providing a photodetecting means for monitoring an intensity of the laser light taken out by a light-splitting means. CONSTITUTION:A laser beam 15 from a laser oscillator 1 enters an adjuster 2 adjusting an amount of light, and then is deflected by a deflecting part 3 so as to scan an image-forming surface L in two dimensions. Further, the beam 15 is split. by a beam splitter (light splitting means) 4. Passing beams are brought into a pupil 5 of an objective lens 6 to intensively scan a sample 8 on a slide glass 7. Meanwhile, beams reflected at the splitter 4 are monitored by a photodetecting means 10 through an aperture 9. An output signal from the photodetecting means 10 is input, to a control part 11, whereby a transmittance of the adjuster 2 is adjusted by the control part 11 so that an amount of light entering the photodetecting means 10 is constant while the light is deflected and scanned. The aperture 9 is placed at an equivalent position to the pupil 5 and therefore, an intensity of the beam cast onto the sample 8 is also constant during the scanning.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laser irradiation device for a laser scanning microscope.

[0002]

2. Description of the Related Art FIG. 3 shows an example of a conventional laser irradiation apparatus for a laser scanning microscope. In FIG. 3, a laser beam 70 emitted from a laser oscillator 50, which is a light source, is linearly reflected by an acousto-optic deflecting element (AOD) 51, which is an optical deflecting element, as shown in FIG. The light is deflected in the original direction, passes through the relay lens 52, the wavelength selection mirror 53, and the relay lens 54 in order, and enters the galvano-type deflection mirror 55. The deflecting mirror 55 is shown in FIG.
In the example shown in (1), it is possible to rotate about an axis parallel to the paper surface. The laser beam deflected by the deflection mirror 55 is
After the image is formed on the image forming plane L by the relay lens 56, the sample 60 on the slide glass is condensed and irradiated by the objective lens 59 through the pupil 58 of the objective lens 59. In addition, as the sample 60, cells and the like which are usually fluorescently stained are used.

The fluorescence from the sample 60 on the slide glass scanned by the two-dimensional laser spot is converted into the objective lens 59.
The laser beam is focused on, travels along the same path as the laser beam, passes through the relay lens 54, is reflected by the wavelength selection mirror 53, and is converted into an electric signal by the fluorescence detector 61. In addition,
In this example, the positions of the pupil 58 of the objective lens 59, the deflecting mirror 55, and the AOD 51 are conjugate with each other. Therefore, the laser beam deflected by the AOD 51 and the deflecting mirror 55 is always incident on the pupil 58 of the objective lens 59. Further, in this example, the acousto-optic deflecting element (AOD) 51 and the galvano-deflecting mirror 55 are used for the two-dimensional deflection scanning of the laser beam.
There is also a laser microscope of a type that performs two-dimensional scanning using an OD, a galvano deflection mirror, a polygon mirror, a resonant deflection mirror, a hologram scanner, or the like.

[0004]

In the conventional laser irradiating device of the laser scanning microscope described above, A
When the OD51 is used, the diffraction efficiency of the laser beam changes depending on the deflection angle as a characteristic of the AOD51, so that there is a problem that the laser intensity applied to the sample 60 on the slide glass changes depending on the scanning position.

FIG. 4 is a diagram showing a laser spot intensity on a slide glass in a conventional example. In FIG. 4, the Z axis indicates the light intensity of the laser spot, the X axis indicates the scanning direction position by AOD, and the Y axis indicates the scanning direction position by the galvano-type deflection mirror. As can be seen from FIG. 4, since the laser spot intensity at the position b is stronger than the position a on the X axis, the fluorescence intensity from the cells near the X = b axis is higher than that at the cells near the X = a axis. There is a problem that the fluorescence becomes strong and the amount of fluorescence emitted by the cell cannot be accurately measured.

Therefore, the present invention has been made in view of the above-mentioned problems, and laser irradiation for making the amount of laser light irradiating a sample in a laser scanning microscope uniform at all positions within the scanning range on the sample surface. The purpose is to provide a device.

[0007]

In order to achieve the above object, a laser irradiation apparatus for a laser scanning microscope according to the present invention comprises a laser oscillator which is a light source, and a laser beam deflecting unit which scans a laser from the laser oscillator. In a laser irradiation device of a laser scanning microscope equipped with an objective lens for converging and irradiating a sample with a scanning laser beam, a light splitting means for taking out a part of the laser beam incident on the objective lens, and a light splitting means for taking out the light by this light splitting means. And a laser light amount adjusting means for adjusting the intensity of the laser beam incident on the objective lens based on the monitoring result by the light detecting means. To do.

[0008]

According to this laser irradiation device, a part of the laser beam incident on the objective lens is taken out by the light splitting means, the intensity of the taken-out laser beam is monitored by the light detecting means, and the monitor output becomes constant. As described above, by adjusting the intensity of the laser beam incident on the light detecting unit, that is, the intensity of the laser beam incident on the objective lens by the laser light amount adjusting unit, the laser beam at all positions within the laser scanning range on the sample surface is adjusted. The strength is constant. Therefore, the measurement accuracy is high, and an accurate image can be obtained.

[0009]

EXAMPLES A laser irradiation apparatus of the present invention will be described below based on examples. FIG. 1 shows the principle of the device of the present invention. In FIG. 1, a laser beam 15 from a laser oscillator 1 is incident on a light quantity adjuster 2 and is then deflected by a laser beam deflecting unit 3 so that an image plane L is two-dimensionally scanned. Further, the laser beam 15 is split by the beam splitter (light splitting means) 4, and the laser beam transmitted through the beam splitter 4 enters the pupil 5 of the objective lens 6,
The objective lens 6 focuses and scans the sample 8 on the slide glass 7.

On the other hand, the laser beam reflected by the beam splitter 4 passes through the opening 9 and is converted into an electric signal by the photo-detecting means 10. The output signal of the light detection means 10 is the control unit 11
The light transmittance of the laser beam of the light amount adjuster 2 is adjusted by the control unit 11 so that the light amount that is input to the light detection unit 10 is constant during the deflection scanning of the laser beam.
However, here, the light amount adjuster 2 and the control unit 11 constitute a laser light amount adjusting means.

Here, the opening 9 has a position equivalent to the pupil 5 of the objective lens 6, that is, the intensity distribution on the surface of the pupil 5 of the laser beam incident on the pupil 5 of the objective lens 6 is on the surface of the opening 9. Since they are arranged at the positions having the same shape except the intensity distribution and the absolute intensity, the intensity of the laser spot irradiating the sample 8 is constant during the scanning. In the above configuration, as the laser oscillator 1 serving as a light source, a gas laser such as a He-Ne laser or an Ar laser, a solid-state laser such as a Ne-YAG laser, a dye laser, a laser diode or the like can be applied. When a semiconductor laser is used as the light source, direct modulation is possible, and the dedicated light quantity adjuster 2 is not needed, so the configuration of the device is simplified. As the light quantity adjuster 2, an acousto-optic adjusting element (AOM), an electro-optical modulating element (EOM), an optical modulating element using liquid crystal, or the like can be applied. As the laser beam deflection unit 3, an AOD, a polygon mirror, a galvano mirror, a holographic element, a resonant mirror, or the like can be applied.

Further, a plate beam splitter, a cubic prism type beam splitter, a pellicle or the like can be applied as the beam splitter 4, that is, the light splitting means.
As the light detection means 10, a silicon photodiode,
A semiconductor photodetector element such as an avalanche photodiode is inexpensive and desirable. Further, although the size of the aperture 9 is not particularly limited in FIG. 1, when the diameter of the laser beam incident on the pupil 5 of the objective lens 6 is larger than the size of the pupil 5,
The size of the opening 9 is preferably equivalent to the size of the pupil 5 of the objective lens 6. Further, it is desirable that the opening 9 and the detection surface of the light detection means 10 are in close contact or as close as possible.

Next, a more specific embodiment will be described with reference to FIG. However, in this example, fluorescently stained cells are used as the sample. In FIG. 2, a laser beam 47 from a laser oscillator (an air-cooled Ar laser in this embodiment) 20 is incident on a beam expander 23 via an acousto-optic adjusting element 22 as a light quantity adjuster, and the beam diameter becomes an appropriate size. Expanded. Further, the laser beam 47 includes a cylindrical lens 24, an acoustic light deflecting element 25, a correction cylindrical lens 26, a cylindrical lens 27,
A one-dimensional deflection optical system composed of a spherical lens 28 and a spherical lens 28 condenses and one-dimensionally scans in the direction of arrow m in FIG.
Subsequently, the laser beam 47 passes through the wavelength selection mirror 29 and the relay lens 30, and the galvano deflection mirror 31.
Incident on.

The deflecting mirror 31 is connected to the galvanometer 32 and can be rotated in the direction of arrow p by driving the galvanometer 32. The laser beam deflected and scanned by the deflecting mirror 31 is imaged on the image plane L by the relay lens 33.
After being focused on the image, it is incident on a beam splitter (a prism beam splitter in this embodiment) 34. In this embodiment, the beam splitter 34 transmits 80% of the laser beam incident on it. The component of the laser beam that has passed through the beam splitter 34 enters the pupil 35 of the objective lens 36, and is focused and scanned by the objective lens 36 onto the sample 38 on the slide glass 37.

On the other hand, the laser beam reflected by the beam splitter 34 passes through the opening 39 and is then passed through the optical attenuation filter 4.
After the amount of light is attenuated by 0, the light is incident on the light detecting means (a silicon photodiode in this embodiment) 41 and converted into an electric signal. The output electric signal of the silicon photodiode 41 is input to the control unit 42. The control unit 42 adjusts the laser beam transmittance of the acousto-optic adjusting element 22 so that the amount of light incident on the silicon photodiode 41, that is, the intensity of the laser spot focused on the sample 38 becomes constant during scanning. Controlled. In this embodiment, the laser spot scans the sample 38 two-dimensionally.

The fluorescence emitted from the sample 38 is condensed by the objective lens 36, and the component transmitted through the beam splitter 34 forms an image on the image plane L, is reflected by the deflecting mirror 31, and is emitted by the wavelength selection mirror 29. The components are selectively transmitted, and the transmitted fluorescence component is converted into an electric signal by the fluorescence detector 43.
In this embodiment, the fluorescence component is one-dimensionally scanned on the fluorescence detector 43 in the direction of arrow n in FIG.

The output electric signal from the fluorescence detector 43 is stored in a memory of a data processing device (not shown) in correspondence with the scanning position of the laser spot, and after data processing is displayed as a fluorescence image on a CRT or the like. In the configuration shown in FIG. 2, the pupil 35 of the objective lens 36 and the galvano-deflecting mirror 31 are at positions conjugate with each other with respect to the relay lens 33, and the deflecting mirror 31 and the acoustic light deflecting element 25 are also
The collimating lenses 28 and 30 are at positions conjugate with each other. Therefore, the position of the laser beam passing through the acoustic light deflecting element 25, the galvano-deflecting mirror 31, and the pupil 35 of the objective lens 36 does not move even if the laser beam is scanned.

Further, in this embodiment, of the fluorescence collected by the objective lens 36, the component reflected by the beam splitter 34 is transmitted through the filter 44 for cutting the laser beam reflected from the sample 38 and is passed to the eyepiece lens 45. It is used for observing a fluorescent image with the naked eye after incidence. Of course, the fluorescence image may be observed with a TV camera or the like instead of the naked eye, and the beam splitter 34 may have wavelength selectivity.

In the above embodiment, the fluorescence signal is one-dimensionally scanned on the fluorescence detector 43. However, the detection surface of the fluorescence detector 43 is provided with a slit for passing only the one-dimensionally scanned fluorescence signal. Thus, when a one-dimensional confocal laser scanning microscope is used, a higher resolution fluorescence image can be obtained.

[0020]

Since the laser irradiation apparatus of the present invention includes the light splitting means, the light detecting means, and the laser light amount adjusting means as described above, it has the following effects. (1) Since the intensity of the laser spot applied to the sample is constant irrespective of the scanning position, when observing the fluorescently stained sample, a fluorescent image of the original intensity distribution of the sample can be obtained. (2) Since the observation surface of the sample is scanned with the same laser spot intensity, the fluorescence image of the sample accurately reflects the original fluorescence intensity of the sample. Therefore, the measurement of the standard sample for correcting the non-uniformity of laser irradiation and the correction after the fluorescence image acquisition are not necessary, and the time to obtain the final image is shortened. (3) DN contained in individual cells based on the fluorescence image of the cells
When measuring the fluorescence amount of the sample such as obtaining the A amount, a highly accurate measurement result can be obtained. (4) Even when the fluorescent dye is discolored depending on the laser excitation intensity or the fluorescence intensity is saturated with respect to the laser excitation, the laser irradiation intensity is constant within the scanning range. An image that accurately reflects the distribution can be obtained.

[Brief description of drawings]

FIG. 1 is a diagram for explaining the principle of a laser irradiation apparatus of the present invention.

FIG. 2 is a configuration diagram of a laser irradiation apparatus according to an embodiment.

FIG. 3 is a configuration diagram of a laser irradiation apparatus according to a conventional example.

FIG. 4 is a diagram showing an intensity distribution of a scanning laser spot on a sample in a conventional laser irradiation device.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 laser oscillator 2 light quantity adjuster 3 laser beam deflector 4 beam splitter (light splitting means) 5 pupil of objective lens 6 objective lens 8 sample 9 aperture 10 light detecting means 11 control section

Claims (3)

[Claims] 1. A laser irradiation apparatus for a laser scanning microscope, comprising a laser oscillator as a light source, a laser beam deflector for scanning a laser from the laser oscillator, and an objective lens for converging and irradiating a sample with a scanning laser beam. In, a light splitting means for extracting a part of the laser beam incident on the objective lens, and a light detecting means for monitoring the intensity of the laser beam extracted by the light splitting means,
A laser irradiation device for a laser scanning microscope, comprising: a laser light amount adjusting means for adjusting the intensity of a laser beam incident on the objective lens based on a monitoring result by the light detecting means. 2. The laser irradiation apparatus for a laser scanning microscope according to claim 1, wherein the light splitting means is arranged between the laser beam deflector and the pupil of the objective lens. 3. The method according to claim 1, wherein an opening is provided at a position equivalent to a pupil of the objective lens, and the opening is arranged between the light splitting means and the light detecting means.
A laser irradiation device of the laser scanning microscope described.