JPH11174332A - Laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser microscope capable of selectively monitoring only illumination wavelength being an object. SOLUTION: Since laser power after light control is monitored by a reference light separation mirror 4 provided astern of dimming filters 3 and 23, correlation between the laser power illuminating a sample SP and output from an intensity detector 17 for monitoring light quantity is obtained. Since a laser shutter 5 is arranged at the rear of the mirror 4, the laser power is monitored in a state where the shutter 5 is closed and the laser power of the laser beam for illumination is adjusted by the filters 3 and 23. In the case of simultaneously monitoring the laser power having plural kinds of wavelength from plural laser beam sources 1 and 21, the wavelength of reference light selected by a wavelength selection device 16 is successively switched in proper timing. The selected wavelength is monitored by a control processor 14 together with output from the detector 17.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a laser microscope for observing a sample image illuminated with laser light, and more particularly to a laser microscope capable of accurately monitoring the power of laser light from a light source.

[0002]

2. Description of the Related Art As a laser microscope, for example, there is a laser scanning microscope which detects a sample image by scanning a laser beam on a sample. Such a laser scanning microscope generally includes a scanning unit that appropriately scans a sample with a spot-shaped laser beam, a device that detects reflected light and fluorescence from the sample, and displays the detected light on a monitor as an image of the sample. And a device for performing the operation.

When measuring reflected light, fluorescence, etc. from a sample using this laser scanning microscope, the conditions for measurement are kept constant, and only specific parameters are changed to carry out comparative experiments, or the same conditions are used. And re-measurement and re-testing are performed. In such a case, the measurement conditions,
The situation must be precisely controlled. Among these conditions, a change in the laser power, in particular, directly leads to a change in the brightness of the detected image. Therefore, accurate control of the laser power is an important issue in performing reproducible measurement.

[0004] Laser power is controlled by utilizing the power control function of the laser tube itself or by inserting an optical member for changing the transmittance into the illumination optical path. In the case of a laser having no laser power control function, such as a He-Ne laser, the laser power is controlled only by the above-described method. Also,
In the method of (1), when the laser power is controlled continuously, an optical member that changes the transmittance, such as a continuous ND filter or an acousto-optic device, is used as the optical member that changes the transmittance.

When the laser power is controlled by the above method, it is necessary to monitor the laser light incident on the sample in order to maintain the reproducibility of the laser power. At this time, in order to accurately set the laser power for illuminating the sample, a mirror or the like is appropriately inserted into the illumination light path, and the laser light is temporarily taken out of the illumination light path and monitored.

[0006]

In the method of directly monitoring the laser light for illumination as described above, when laser lights of a plurality of wavelengths are mixed, the total power of the mixed laser light is monitored. Will be. Therefore, when controlling the laser power for the laser light of a single wavelength, it is necessary to stop the oscillation of another laser device or to shut off the laser light from the other laser device with a shutter for each laser device.

However, the method of stopping the oscillation of another laser device as described above requires a considerable time from power-on to stabilization depending on the type of the laser device. The problem is that powering down is usually undesirable. In the latter method of blocking laser light from another laser device, it is necessary to separately provide shutters by the number of laser devices provided corresponding to each wavelength, resulting in an increase in size of the device. There is a problem.

Further, in a multi-wavelength oscillation laser such as a Kr-Ar laser which emits laser light of a plurality of wavelengths from one laser, it was not simply possible to monitor the laser power of a single wavelength.

Furthermore, in the method of separating and monitoring the illumination light as described above, the laser power monitoring operation must be completed before the actual measurement, and the laser power does not change during the measurement. I had to believe and do it.

Accordingly, an object of the present invention is to provide a laser microscope capable of selectively monitoring only illumination light of a target wavelength.

Another object of the present invention is to provide a laser microscope capable of monitoring laser power at an appropriate timing without affecting laser light illuminating a sample.

[0012]

In order to solve the above problems, a laser microscope according to the present invention comprises a light source for generating a plurality of laser lights having different wavelengths from each other, and an objective lens for transmitting the plurality of laser lights from the light source. An illumination optical system that irradiates the sample with the light, an observation optical system that acquires light emitted from the sample by irradiating the plurality of laser lights to obtain a sample image, and the plurality of laser lights from the light source is applied to the sample. A splitting device that splits a part of the plurality of laser lights as reference light before being irradiated, a wavelength selecting device that selects light of a predetermined wavelength component among the reference lights split by the splitting device, An intensity detection device for detecting the intensity of the light of the predetermined wavelength component selected by the device.

In a preferred aspect, the apparatus further comprises a shutter device disposed on an optical path between the splitting device and the sample and capable of blocking irradiation of the sample with the plurality of laser beams. .

In another aspect of the present invention, there is provided a laser microscope, comprising: a light source for generating laser light; an illumination optical system for irradiating the sample with the laser light from the light source via an objective lens; An observation optical system that obtains a sample image by acquiring light emitted from a sample, a splitting device that splits a part of the laser light as reference light before the laser light from the light source is applied to the sample, and the splitting device. An intensity detection device that detects the intensity of the reference light split by the device, and a shutter device that is disposed on an optical path between the splitting device and the sample and that can block irradiation of the sample with the laser light. It is characterized by having.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of a laser microscope according to the present invention will be described below with reference to the drawings.

FIG. 1 is a block diagram of a laser scanning microscope for exciting a sample at a plurality of wavelengths. In the figure,
Only the main members of the optical system constituting the laser scanning microscope are described, and the reduction and enlargement optical members are omitted.

As is apparent from the figure, the laser scanning microscope has a pair of laser light sources 1 and 21 for generating laser beams of a plurality of wavelengths for illumination, and a laser beam emitted from each of the laser light sources 1 and 21 as a light source device. A pair of excitation filter devices 2 and 22 for respectively selecting a laser beam having a wavelength optimal for illumination from light, and a pair of dimming filter devices 3 for adjusting the power of the laser beam passing through each of the excitation filter devices 2 and 22; 23, and a reflection mirror 24 for mixing laser beams of a plurality of wavelengths that have passed through each of the dimming filter devices 3 and 23
And a dichroic mirror 25.

In the laser scanning microscope, a reference light separating mirror 4 serving as a splitting device for splitting a part of a plurality of wavelengths of laser light emitted from the light source device as a reference light, as an illumination light monitoring device, A wavelength selection device 16 that selects and transmits only reference light of a target wavelength from the reference light partially split by the separation mirror 4, and intensity detection that detects the power of the reference light selected by the wavelength selection device 16. The device 17 is provided.

The laser scanning microscope, as a scanning detector, uses a detection light separating mirror 6 for separating laser light for illumination and detection light, and scans an optical path of the laser light reflected by the detection light separation mirror 6. The horizontal scanner 7 and the vertical scanner 8 for adjusting the laser beam, and the laser beam passing through the horizontal scanner 7 and the vertical
The objective lens 9 condensed at the position P, and the laser light is emitted from the sample SP, passes through the objective lens 9, is scanned by the vertical scanner 8 and the horizontal scanner 7, and passes through the detection light separating mirror 6. A pinhole device 11 for selectively passing light, a detection filter device 12 for selecting light of a desired wavelength from the detection light passing through the pinhole device 11, and a detection light passing through the detection filter device 12 is converted into an electric signal. And a photoelectric conversion device 13.

Further, the laser scanning microscope is disposed between the reference light separating mirror 4 and the detection light separating mirror 6 to block the laser light from the light source device and prevent the laser light from being incident on the sample SP. And a laser shutter 5 that can be used.

Further, the laser scanning microscope controls the entire laser scanning microscope and controls the photoelectric conversion device 13.
The electric signal detected in step 15 is subjected to appropriate processing to
And a control processing device 14 for displaying a sample image on the display.

The control processing device 14 includes the laser light source 1,
21 to control the laser power output from them, to control the excitation filter devices 2 and 22 to select the wavelength of the laser light used for illumination, and to control the dimming filter devices 3 and 23. To adjust the intensity of the laser light used for illumination. Further, the control processing device 14 controls the wavelength selection device 16 to select the wavelength of the laser light to be detected as the reference light, and based on the detection output of the intensity detector 17, the reference light at the selected wavelength. Monitor the power of. Further, the control processing device 14 controls the scanners 7 and 8 to monitor the scanning spot of the excitation laser beam at the position of the sample SP, and controls the stage 10 to monitor the position of the sample SP. By controlling 11, a pinhole having a required aperture diameter is arranged on the optical path of the detection light, and the detection filter device 12 is controlled to select only the required detection wavelength and make it incident on the photoelectric conversion device 13.

The excitation filter devices 2, 2 provided in the light source device
Numeral 2 is a turret plate having a plurality of band-pass filters fixed in place, for example, and the turret plate is electrically rotated to place any one of the band-pass filters on the optical path of the laser light. , 21 can be extracted only from a laser beam having a desired wavelength.

Each of the dimming filter devices 3 and 23 has a dimming function in the laser light sources 1 and 21 (for example, He-N).
e-laser) to finely adjust the laser power in an analog manner. As the dimming filter devices 3 and 23, for example, a continuous ND filter in which the transmittance continuously changes, or the transmittance changes continuously by changing the amplitude of the compression wave in the crystal using an acousto-optic effect. Such an acousto-optic element can be used, and the laser power of the laser beam output from the light source device can be easily adjusted based on a control signal from the control processing device 14.

The reference light separating mirror 4 provided in the illumination light monitoring device is for directly monitoring the light amount of the laser light supplied from the light source device, and may be, for example, a simple glass plate. By such surface reflection of the plate glass, several percent of the laser light is split and guided to the wavelength selection device 16 and the intensity detector 17 as reference light. Such a plate glass has almost no wavelength selectivity, and the divided reference light contains a plurality of wavelengths of laser light having an intensity corresponding to the wavelength distribution of the laser light emitted from the light source device.

The wavelength selecting device 26 includes a reference light separating mirror 4
Target wavelength is selected from the reference light divided by the above. The wavelength selecting device 16 can switch the wavelength to be selected based on a control signal from the control processing device 14. For switching the wavelength, various mechanisms can be employed depending on the required switching speed. If you do n’t need to switch very quickly,
For example, the objective can be achieved by manually inserting a desired bandpass filter into a positioning holder. If a rather quick switching is required, the object can be achieved by electrically rotating a turret plate to which a plurality of bandpass filters are attached and disposing a desired bandpass filter on the optical path of the reference light. If higher speed is required, it is also possible to select a wavelength with an acousto-optic conversion element (AOTF) and to guide only the reference light of the required wavelength to the intensity detector 17.

The intensity detector 17 is composed of, for example, a silicon photodiode (SPD), and converts an output current into a voltage according to the power of the reference light. This allows the control processor 14 to monitor the laser power of the laser beam supplied to the sample SP as a voltage.

The laser shutter 5 moves on the optical path of the laser light for illumination to prevent the laser light from being incident on the sample SP, and the laser shutter 5 moves out of the optical path of the laser light for illumination and moves the laser light. The laser beam is movable between a retracted position that allows the laser beam to enter the sample SP, and is controlled by the control processing device 14 so that the laser beam is incident on the sample SP only when necessary.

The detection light separating mirror 6 separates the laser light for illumination and the detection light. For example, a dichroic mirror is used for fluorescence observation, and a half mirror or a deflection beam splitter is used for reflection observation. I do.

As the horizontal scanner 7 and the vertical scanner 8, for example, a galvanometer mirror can be used.

The pinhole device 11 is composed of, for example, a plate provided with a plurality of pinholes having different opening diameters at appropriate positions. The plate is electrically rotated to place one of the pinholes on the optical path of the detection light. Thus, a necessary confocal effect can be obtained.

The detection filter device 12 comprises, for example, a turret plate in which a plurality of bandpass filters are fixed in place, and this turret plate is rotated electrically to arrange any of the bandpass filters on the optical path of the detection light. Thereby, it is possible to extract only light having a desired wavelength from the fluorescence and reflected light from the sample SP.

The photoelectric conversion device 13 is composed of, for example, a photomultiplier tube (PMT) and outputs a signal corresponding to the intensity of light emitted from the sample.

The operation of the apparatus shown in FIG. 1 will be described below. After the laser light emitted from the laser light sources 1 and 21 has its wavelength selected by the excitation filters 2 and 22, the power is adjusted by the dimming filters 3 and 23. The dimmed laser light passes through the reference light separation mirror 4, is reflected by the detection light separation mirror 6 that separates the illumination laser light and the detection light, and is guided to the horizontal scanner 7 and the vertical scanner 8. The laser light incident on both scanners 7 and 8 becomes a point light source by the objective lens 9 and two-dimensionally scans the sample SP. On the other hand, the detection light (fluorescence in the case of fluorescence observation, reflected light in the case of reflection observation) from the sample SP generated by this laser light is reversed in the optical path, scanned by the vertical scanner 8 and the horizontal scanner 7, and detected. The light passes through the light separating mirror 6 and is separated from the irradiation laser light. The detection light transmitted through the detection light separating mirror 6 enters the photoelectric conversion device 13 and is converted into an electric signal, and is converted into an image by the control processing device 14.

At this time, since the reference light separating mirror 4 provided behind the light control filters 3 and 23 can monitor the laser power of the laser light after the light control, the laser power for illuminating the sample SP and the light amount monitor. Intensity detector 17
Will be correlated with the output of Conversely, if the dimming filters 3 and 23 are adjusted so that the output values of the intensity detector 17 become the same, it is easy to make the laser power illuminating the sample SP the same. Further, since the laser shutter 5 is disposed behind the reference light separating mirror 4, the laser shutter 5 is closed, that is,
The laser power can be monitored while the sample SP is not being irradiated with the laser light, and the dimming filters 3 and 23 can appropriately adjust the laser power of the laser light for illumination.

When simultaneously monitoring the laser power of a plurality of wavelengths from the plurality of laser light sources 1 and 21, the wavelength of the reference light selected by the wavelength selection device 26 is sequentially switched at an appropriate timing. The selected wavelength is the intensity detector 17
Is monitored by the control processor 14 together with the output of
The laser power for each wavelength can be obtained. At this time, if the transmission and reflection wavelength characteristics of the reference light separation mirror 4, the wavelength selection device 16, the dichroic mirror 25, and the like are measured in advance, the absolute value of the intensity of the illumination laser light on the sample SP is roughly estimated. You can also. By providing the illumination light monitoring device as described above, it is not necessary to provide a special monitoring mechanism for each of the laser beams having a plurality of wavelengths.

Although the present invention has been described with reference to the embodiment, the present invention is not limited to the above embodiment. For example, the laser devices 1 and 21 can be a single laser device (for example, a Kr-Ar laser) that generates laser light of a plurality of wavelengths. In this case, power monitoring at a single wavelength that is not provided in the laser device itself is possible. When a single laser device is used, a single excitation filter device and a single light control filter device are sufficient.

In the above embodiment, the scanning laser microscope has been described. However, even a non-scanning laser microscope that illuminates the entire observation area of the sample SP may emit the laser light for illumination as described above. Monitoring can be performed by an illumination light monitoring device including the reference light separation mirror 4, the wavelength selection device 16, the intensity detector 17, and the like. In this case, the scanners 7 and 8 are not used.

In the above embodiment, the confocal type laser microscope has been described, but the present invention is not limited to the confocal type laser microscope. In this case, the pinhole device 11 is not used.

[0040]

According to the laser microscope of the present invention, before the sample is irradiated with the plurality of laser beams from the light source, the splitting device splits a part of the sample as reference light, and the wavelength selecting device The light of the predetermined wavelength component is selected from the reference lights split by the splitting device, and the intensity detection device detects the intensity of the light of the predetermined wavelength component selected by the wavelength selection device. Only a laser beam having a desired wavelength can be selectively monitored by a simple mechanism.

According to a preferred aspect, the apparatus further comprises a shutter device disposed on an optical path between the splitting device and the sample and capable of blocking irradiation of the plurality of laser beams to the sample. Only laser light of the target wavelength can be selectively monitored while the irradiation of the laser light to the sample is cut off, and damage to the sample can be easily avoided.

According to another aspect of the laser microscope,
The intensity detection device detects the intensity of the reference light split by the splitting device, the shutter device is disposed on an optical path between the splitting device and the sample, the laser to the sample Since light irradiation can be blocked, laser power can be accurately monitored at necessary timing while avoiding damage to the sample.

[Brief description of the drawings]

FIG. 1 is a block diagram illustrating a structure of a laser scanning microscope according to an embodiment.

[Explanation of symbols]

 Reference numerals 1, 21 Laser light source 2, 22 Excitation filter device 3, 23 Dimming filter device 4 Reference light separation mirror 5 Laser shutter 6 Detection light separation mirror 7 Horizontal scanner 8 Vertical scanner 9 Objective lens 10 Stage 11 Pinhole device 12 Detection filter 13 Photoelectric converter 14 Control processor 16 Wavelength selector 17 Intensity detector 24 Total reflection mirror 25 Dichroic mirror SP Sample

Claims (3)

[Claims] 1. A light source for generating a plurality of laser beams having different wavelengths from each other, an illumination optical system for irradiating a sample with the plurality of laser beams from the light source via an objective lens, and irradiating the plurality of laser beams An observation optical system that acquires light emitted from the sample to obtain a sample image, and divides a part of the plurality of laser lights as reference light before the plurality of laser lights from the light source are irradiated on the sample. A splitting device; a wavelength selecting device that selects light of a predetermined wavelength component among the reference lights split by the splitting device; and an intensity detection that detects the intensity of the light of the predetermined wavelength component selected by the wavelength selecting device. And a device. 2. The apparatus according to claim 1, further comprising a shutter device disposed on an optical path between the splitting device and the sample and capable of blocking irradiation of the plurality of laser beams to the sample. Laser microscope. 3. A light source for generating a laser beam, an illumination optical system for irradiating the sample with the laser beam from the light source via an objective lens, and acquiring light emitted from the sample by the irradiation of the laser beam. An observation optical system that obtains a sample image; a splitting device that splits a part of the laser light as reference light before the laser light from the light source is applied to the sample; and the reference light split by the splitting device. A laser, comprising: an intensity detection device that detects the intensity of the laser beam; and a shutter device that is disposed on an optical path between the splitting device and the sample and that can block irradiation of the sample with the laser light. microscope.