JPH11249022A - Scanning type laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a scanning type laser microscope constituted so that a focusing position can be easily and quickly obtained. SOLUTION: The scanning speed of a laser beam with respect to a sample 9 by an X-direction scanner 4 and a Y-direction scanner 5 cap be switched by a scanning controller 17. When the sample 9 is set at a non-focusing position, the scanning speed of the laser beam is switched to be higher in comparison with a case that the sample 9 is set at the focusing position. By such high-speed control action, the focusing position of the sample 9 is set while changing a relative distance between the sample 9 and an objective lens 8 based on a detection signal with respect to the sample 9.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning laser microscope which irradiates a sample with a laser beam while scanning the same, and obtains image information by detecting light from the sample.

[0002]

2. Description of the Related Art A scanning laser microscope irradiates a point light source such as a laser beam through an objective lens while scanning the sample in the X-axis and Y-axis directions, and again emits fluorescence or reflected light from the sample through an objective lens. Detected by a photodetector via an optical system, 2
By obtaining the density information of a two-dimensional image, and displaying the two-dimensional distribution of the density information on an image output device such as a CRT monitor or a color printer in correspondence with the XY scanning position of the point light source, It can be observed as image information.

[0003] Further, such a scanning laser microscope is provided at a position conjugate with a sample of a detection optical system having a detector.
There is also known a confocal scanning laser microscope in which a stop having a diameter approximately equal to the diffraction limit of illumination light or measured light is provided so that only information on a focused surface can be detected.

In such a confocal scanning laser microscope, since only the information on the focal plane can be detected, a tomographic image, that is, three-dimensional information can be obtained optically without damaging the sample. By cutting the information on the focal plane, a very sharp image can be obtained.

[0005] In a general optical microscope, it is possible to roughly determine whether the target sample position is in the vertical direction from the current focal position, based on a change in information on the non-focus plane. For example, JP-A-59-182409
The optical microscopes disclosed in Japanese Patent Application Laid-Open No. HEI 64-6416 and Japanese Patent Application Laid-Open No. Sho 64-42416 are equipped with an autofocus function for quickly focusing using the defocus amount, that is, information from an unfocused surface. The focus position can be obtained.

[0006]

However, in the above-mentioned confocal scanning laser microscope, since information on an unfocused surface cannot be obtained as in an optical microscope, it is difficult to move a target sample to a focused position. However, there is a problem that it requires operator experience and work time.

For this reason, even if an autofocus function based on the same principle as that of the above-described optical microscope is to be mounted on a confocal microscope, the confocal scanning laser microscope cannot obtain information on an out-of-focus plane. Can not do it.

Further, in such a confocal scanning laser microscope, two-dimensional information is imaged by scanning a point light source on a sample surface, so that it takes one second to several seconds to obtain one image. It takes a lot of time and makes it difficult to find the in-focus position.

Therefore, conventionally, in order to make it possible to easily search for the in-focus position, a method of reducing the number of scans per screen to shorten the time required to acquire one image is considered. Have been. However, in the method used so far, since the position of the sample is adjusted while displaying the image acquired by scanning,
There are problems that the operation time is not sufficiently shortened, and that the sample is unnecessarily irradiated with light, thereby damaging the sample. The present invention has been made in view of the above circumstances, and has as its object to provide a scanning laser microscope capable of easily and quickly obtaining a focus position.

[0010]

Means for Solving the Problems In general, a galvanometer mirror (hereinafter, galvanometer) is used as a scanner used in a scanning laser microscope. In consideration of the linearity of the galvano scanning, it is required to drive at a low repetition frequency of several hundred Hz or less. However, to obtain an image having several hundreds to several thousands of scanning lines, the above-described method is used. Thus, a time of one second to several seconds is required. However, when the linearity of scanning is ignored, the galvanometer can respond to the drive signal at a frequency several times to about ten times that of a normal image acquisition. Also, if the sample is at the in-focus position,
When the sample is at the out-of-focus position while a large measurement signal is obtained, the measurement signal is absent.

Focusing on the above points, the present invention is configured as follows. According to a first aspect of the present invention, there is provided a scanning laser microscope in which a sample is irradiated with a laser beam while being scanned, and image information is obtained from a detection signal corresponding to a light amount from the sample. And a scanning speed control means for switching the scanning speed of the point light source at a higher speed when the sample is at the out-of-focus position than when the sample is at the in-focus position. Under the high-speed control of the scanning speed control means, the in-focus position of the sample is set based on the detection signal for the sample.

According to a second aspect of the present invention, in the first aspect, there is further provided a filter means for obtaining an average light amount within a scanning range as a detection signal from the sample.
According to a third aspect of the present invention, in the first aspect, a focus position detecting means for detecting a focus position of the sample from a detection signal from the sample, and a detection output of the focus position detection means. A focus position adjusting means for adjusting a focus position with respect to the sample.

As a result, according to the first aspect of the present invention,
Since a detection signal from the sample can be obtained with a faster update time than in the normal image acquisition, a quick focusing operation can be performed, and the illumination time of unnecessary light on the sample can be shortened.

According to the second aspect of the present invention, an average amount of light in the scanning range on the sample can be obtained as a detection signal, so that an accurate focusing operation can be performed. According to the invention described in claim 3, it is possible to automate all focusing operations that require the experience of the operator.

[0015]

Embodiments of the present invention will be described below with reference to the drawings. (First Embodiment) FIG. 1 shows a schematic configuration of a confocal scanning laser microscope to which the present invention is applied.

In this case, the laser beam (illumination light) from the laser oscillator 1 passes through the beam expander 2 and is reflected by an optical path splitting element 3 such as a beam splitter or a dichroic mirror. The laser beam emitted from the Y-direction scanner 5 is made incident on the objective lens 8 via the pupil projection lens 6 and the imaging lens 7 so that the surface of the sample 9 is Dimensional scanning is performed.

Then, the fluorescent light (or reflected light) from the sample 9 is returned to the optical path splitting element 3 by following the same optical path as the laser beam, and then transmitted through the optical path splitting element 3 and the confocal optical system 10 to obtain dichroic light. Light is received by the photoelectric conversion circuit 12 via a wavelength selection element 11 such as a mirror or an interference filter.

Here, for simplicity, the photoelectric conversion circuit 12
Shows a case where a plurality of wavelength selection elements 11
The present invention can also be applied to a case where a plurality of fluorescent wavelengths can be measured by the photoelectric conversion circuit 12 and the photoelectric conversion circuit 12.

The photoelectric conversion circuit 12 converts the fluorescence (or reflected light) from the sample 9 received through the wavelength selection element 11 into an electric signal indicating luminance information, and converts the luminance information through a low-pass filter 13. A / D conversion circuit 14 converts the data into digital data,
Is input as a detection signal. Here, as the low-pass filter 13, a filter having a cutoff frequency equal to or lower than the frequency of the driving waveform of the X-direction scanner 4 to be described later and higher than the frequency of the driving waveform of the Y-direction scanner 5 is used.

The data processing device 15 includes the A / D conversion circuit 1
After processing the digital data from the CRT 4, image information is displayed on the CRT monitor 16. Further, the data processing device 15 gives a command to the scanning control device 17 according to a change in the luminance information given from the A / D conversion circuit 14 when the sample 9 is at the focal position and when the sample 9 is at the non-focus position, respectively. The operation settings of the scanning drive circuits 18 and 19 are controlled. Here, the scan driving circuit 18
The scanning drive circuit 19 is for driving the X-direction scanner 4, and is for driving the Y-direction scanner 19.

Next, the operation of the embodiment configured as described above will be described. First, a case where a normal image is acquired in a state where the sample 9 is arranged at a focused position will be described.

In this case, in accordance with an instruction from the scanning control device 17, the scanning driving circuit 18 is provided with a signal as shown in FIG.
Waveform data for outputting a drive waveform of the X-direction scanner 4 of about 00 Hz is written, and at the same time, waveform data for outputting a drive waveform of the Y-direction scanner 5 as shown in FIG. It is.

In this state, when a laser beam is emitted from the laser oscillator 1,
The light is reflected by the optical path splitting element 3 and is
It is scanned two-dimensionally by the direction scanner 5 and passes through the pupil projection lens 6, the imaging lens 7 and the objective lens 8, and is scanned on the surface of the sample 9.
Dimensionally scanned.

In this case, the scanning with the sample 9 by the X-direction scanner 4 and the Y-direction scanner 5 is performed by sequentially scanning the scanning lines in one direction from the upper side to the lower side of the surface of the sample 9 as shown in FIG. It will be scanned.

In this state, the fluorescent light (or reflected light) from the sample 9 follows the same optical path as the laser beam and returns to the optical path splitting element 3, where the optical path splitting element 3, the confocal optical system 10,
Further, the light is received by the photoelectric conversion circuit 12 via the wavelength selection element 11, converted into an electric signal indicating luminance information synchronized with the above-described XY scanning, and further converted by the A / D conversion circuit 14 via the low-pass filter 13. Converted to digital data,
After being processed as two-dimensional image information by the data processing device 15, it is displayed on the CRT monitor 16.

Such a series of operations is performed under the condition that the sample 9 is in focus, that is, focused, and the A / D conversion circuit 14 outputs the luminance information of the sample 9 to the output data. Is output by the data processing device 15 as C
Image information is displayed on the RT monitor 16.

However, when the sample 9 is out of focus, that is, at the out-of-focus position, the A / D conversion circuit 14
No input is made, and accordingly, no image is displayed on the CRT monitor 16.

When the operator recognizes that the image is no longer displayed, that is, that the image is at the out-of-focus position, the operator instructs the scan control circuit 17 to the scan drive circuit 18 as compared with the case of the normal image acquisition described above. In this case, drive data having a sufficiently high repetition frequency, here, waveform data for outputting the drive waveform of the X-direction scanner 4 of about 1 KHz shown in FIG. An operation of writing waveform data for outputting a driving waveform of the Y-direction scanner 5 shown in FIG. Further, the non-input state may be automatically recognized by the data processing device 15, and the above instruction may be given to the scanning operation control device 17. In this case, it is desirable to increase the repetition frequency of the drive waveform of the X-direction scanner 4 by using a highly symmetrical waveform such as a sine wave in consideration of galvano characteristics.

In this state, when high-speed scanning of the X-direction scanner 4 and the Y-direction scanner 5 is started by the scanning driving circuits 18 and 19, the laser beam from the laser light source 1 is applied to the surface of the sample 9 as shown in FIG. Are scanned two-dimensionally, and an electric signal from the photoelectric conversion circuit 12 is input to the A / D conversion circuit 14 accordingly.

In this case, the input side of the A / D conversion circuit 14 has a cut-off frequency equal to or lower than the frequency of the driving waveform of the X-direction scanner 4 and higher than the frequency of the driving waveform of the Y-direction scanner 5. With the provision of the low-pass filter 13, the output data from the A / D conversion circuit 14 represents the average amount of light in one X-direction scanning line or one Y-direction scanning frame.

Then, the data processor 15 displays the temporal change of the data indicating the average light amount in one line or one frame on the CRT monitor 16 as, for example, a bar graph or a line graph.

The operator refers to the contents of these graphs and moves the stage on which the sample 9 is mounted or the objective lens 8 in the optical axis direction, and performs a focusing operation while changing the relative distance between the sample 9 and the objective lens 8. Do. Then, when the focus position is reached, A
When the input to the / D conversion circuit 14 rises, A at this time
From the change in the graph displayed on the CRT monitor 16 as the output data of the / D conversion circuit 14 rises,
The focus at which the sample reaches the focal position can be recognized.

From this in-focus state, the scan controller 17 writes waveform data for outputting the drive waveform of the X-direction scanner 4 shown in FIG. By writing the waveform data for outputting the driving waveform of the Y-direction scanner 5 shown in FIG. 2B, the laser beam is two-dimensionally scanned on the sample 9 by the X-direction scanner 4 and the Y-direction scanner 5 again. The monitor 16 displays two-dimensional image information of the sample 9.

Accordingly, this makes it possible to switch the scanning speed of the laser beam with respect to the sample 9 by the X-direction scanner 4 and the Y-direction scanner 5 by the scanning control device 17 and to keep the sample 9 at the out-of-focus position. The scanning speed of the laser beam is switched at a higher speed than when the sample 9 is at the in-focus position. Under this high-speed control, the relative position between the sample 9 and the objective lens 8 is determined based on the detection signal for the sample 9. Since the focus position of the sample 9 is set while changing the distance, a detection signal from the sample 9 can be obtained with an update time that is several to ten times as fast as that of normal image acquisition, and a prompt signal can be obtained. Focusing operation can be performed. In this case, if the scanning density of the X-direction scanner 4 is reduced by increasing the frequency of the driving waveform of the Y-direction scanner 5, the scanning speed can be further increased, so that a more prompt focusing operation can be realized. . As a result, the illumination time of unnecessary light on the sample 9 can be shortened, so that inconvenience such as damaging the sample 9 can be avoided.

Further, by providing a low-pass filter 13 having a cut-off frequency equal to or lower than the frequency of the driving waveform of the X-direction scanner 4 and higher than the frequency of the driving waveform of the Y-direction scanner 5, the detection signal of the sample 9 can be obtained. Is obtained as an average light amount for one line in the X-direction scanning or one frame in the Y-direction scanning, so that a more accurate focusing operation can be performed.

Since the scanning density of the X-direction scanner 4 can be changed arbitrarily by changing the frequency of the driving waveform of the Y-direction scanner 5, the frequency of the driving waveform of the Y-direction scanner 5 can be changed arbitrarily according to the object to be measured. It may be settable. (Second Embodiment) In the first embodiment, the operator performs the focusing operation while referring to the graph indicating the average light amount in one line or one frame.
Some experience was required for operation.

In the second embodiment, the focusing operation is automated. FIG. 5 shows a schematic configuration of the second embodiment of the present invention. The same parts as those in FIG.

In this case, a focal position adjusting means 20 for relatively moving the focal positions of the sample 9 and the objective lens 8 and A / A
A focus position detecting means 21 for judging a focus position from output data from the D conversion circuit 14 is provided, and the focus position adjusting means 20 and the focus position detecting means 21 realize automation of a focusing operation.

In a general microscope, a focusing mechanism is provided for mounting a sample to be observed on a stage and adjusting a relative position between the sample and a focal position of an objective lens. In particular, in an upright microscope, the relative position between the sample and the focal position of the objective lens is adjusted by moving the objective lens up and down. An electric motor or a piezo actuator using a piezoelectric element is generally used.

In the second embodiment, the above-described piezo actuator is used as a drive source for adjusting the relative position between the sample 9 and the focal position of the objective lens 8 in the focal position adjusting means 20. Also, the focus position detecting means 21
Has a function of comparing digital data corresponding to the measurement signal output from the A / D conversion circuit 14 with a preset focus detection level, and according to the measurement signal from the A / D conversion circuit 14, The position at which the acquired data is equal to or higher than the focus detection level is defined as the focus position, and the focus detection signal is used as the focus position adjusting means 2.
0 is output. The focus position detection means 21 is connected to the input side of the A / D conversion circuit 14 as shown by a broken line in the drawing, and detects the focus position from an analog signal input to the A / D conversion circuit 14. Can also be.

In such a configuration, in order to detect the in-focus position, also in this case, a high-speed scanning waveform is instructed from the scanning control device 17 and the X-scanner 4 and the Y-scanner 5 are operated by the scan driving circuits 18 and 19. Start high-speed scanning. Then, the focus position adjusting means 20 first moves in a direction away from the relative distance between the sample 9 and the objective lens 8, and at a predetermined distance, this time, moves the relative distance between the sample 9 and the objective lens 8 at a constant speed. Go closer. The moving speed at this time needs to be appropriately adjusted depending on the resolution in the optical depth (Z) direction due to the confocal effect, the scanning speed of the X scanner 4 and the Y scanner 5, the cutoff frequency of the low-pass filter 14, and the like. However, specifically, while the Y-direction scanner 5 scans for one frame, the resolution is equal to the Z-direction or
A moving speed several times that is desirable.

In this state, when the distance between the sample 9 and the objective lens 8 is short and a part of the sample 9 enters the focal position and the output data of the A / D conversion circuit 14 changes, The focus position detecting means 21 compares data corresponding to the measurement signal of the A / D conversion circuit 14 with a preset focus detection level, and if the data at this time exceeds the focus detection level, the focus position is detected. Is determined as the in-focus position, and a focus detection signal is output to the focus position adjusting means 20. Thereby, the focus position adjusting means 20 stops moving the relative distance between the sample 9 and the objective lens 8 based on the focus detection signal at this time. Also,
The scan control device 17 stops the high-speed scan and starts the scan for normal image acquisition by the focus detection signal. As described in the first embodiment, the CRT monitor 16 The nine surfaces of the sample are displayed.

Accordingly, the focusing operation, which has required the operator's experience to some extent, can be automated, so that a quicker and more reliable focusing operation can be realized. Although the galvanometer mirror has been described in the above-described embodiment, the present invention can be applied to a mirror using an acousto-optic scanner.

[0044]

As described above, according to the present invention, it is possible to obtain a detection signal from a sample with a faster update time than a normal image acquisition, so that a quick focusing operation can be performed. In addition, since the illumination time of unnecessary light on the sample can be shortened, inconvenience such as damaging the sample can be avoided.

Further, since an average light amount in the scanning range on the sample can be obtained as a detection signal, an accurate focusing operation can be performed. Furthermore, since all focusing operations that require the operator's experience can be automated, more prompt and reliable focusing operations can be realized.

[Brief description of the drawings]

FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.

FIG. 2 is a diagram showing driving waveforms of the X and Y direction scanners according to the first embodiment.

FIG. 3 is a diagram illustrating driving waveforms of the X and Y direction scanners according to the first embodiment.

FIG. 4 is a diagram for explaining the operation of the first embodiment;

FIG. 5 is a diagram showing a schematic configuration of a second embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Laser oscillator, 2 ... Beam expander, 3 ... Optical path splitting element, 4 ... X direction scanner, 5 ... Y direction scanner, 6 ... Pupil projection lens, 7 ... Imaging lens, 8 ... Objective lens, 9 ... Sample, Reference Signs List 10: confocal optical system, 11: wavelength selection element, 12: photoelectric conversion circuit, 13: low-pass filter, 14: A / D conversion circuit, 15: data processing device, 16: CRT monitor, 17: scanning control device, 18 , 19: scanning drive circuit, 20: focus position adjusting means, 21: focus position detecting means.

Claims (3)

[Claims] 1. A scanning laser microscope which irradiates a sample with a laser beam while scanning the same and obtains image information from a detection signal corresponding to the amount of light from the sample. And controllable, and when the sample is in the out-of-focus position,
Scanning speed control means for switching the scanning speed of the point light source at a higher speed than when the sample is at the in-focus position, and under the high speed control of the scanning speed control means, based on a detection signal for the sample. A scanning laser microscope, wherein a focus position of the sample is set. 2. The scanning laser microscope according to claim 1, further comprising filter means for obtaining an average amount of light in a scanning range as a detection signal from the sample. 3. A focus position detecting means for detecting a focus position of the sample from a detection signal from the sample, and a focus for adjusting a focus position with respect to the sample in accordance with a detection output of the focus position detection means. The scanning laser microscope according to claim 1, further comprising a position adjusting unit.