JP2006084960A - Three-dimensional confocal microscopic system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To realize a three-dimensional confocal microscopic system capable of three-dimensionally exactly performing the observation of a sample after luminous stimulus by performing three-dimensional image measurement by using a confocal microscope obtained by adding a function for radiating a 2nd laser beam for the luminous stimulus to a Nipkow disk type confocal microscope. <P>SOLUTION: The three-dimensional confocal microscopic system comprises a confocal scanner to acquire the sliced image of the sample as a confocal image through the microscope; a video camera to output the image data of the confocal image; an actuator to move the focal position of the objective of the microscope in an optical axis direction; a control part to generate a scanning waveform signal for making the objective scan in the optical axis direction through the actuator, and an image processing part to form the three-dimensional image of the sample based on the image data acquired by the video camera. It has a laser beam output means connected to the microscope and outputting the laser beam for stimulating the sample, and the sample is stimulated in the optical axis direction by making the objective scan. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a three-dimensional confocal microscope system, and more particularly to a three-dimensional confocal microscope system having a function of irradiating an observation sample with laser light for optical stimulation.

  The confocal microscope can obtain a slice image without making a thin section of the sample, and can construct an accurate three-dimensional stereoscopic image of the sample from the slice image. Therefore, the physiological reaction of living cells in the fields of organisms and biotechnology It is used for observation, morphology observation, or LSI surface observation in the semiconductor market (see, for example, Patent Document 1).

  FIG. 7 is a configuration diagram of the confocal microscope system described in Patent Document 1. The video rate camera 1, the confocal scanner 2, the microscope 3, the actuator 4 and the objective lens 5 are arranged on the same optical axis. The confocal scanner 2 has a nipou disk having a large number of pinholes and a corresponding microlens array, and is a compact add-on type employing a nipou disk system composed of a simple optical system.

  The confocal scanner 2 is attached to the camera port of the microscope 3. The confocal microscope uses a laser beam to input an image of the sample to the confocal scanner 2 via the objective lens 5, the actuator 4 and the microscope 3. The confocal scanner 2 obtains a confocal image of the sample and inputs it to the video rate camera 1.

  FIG. 8 is a time chart of various signals handled by the confocal microscope system of FIG. The video rate camera 1 converts the confocal image into a video signal 101, and inputs the video signal 101 to the confocal scanner 2, the signal input terminal of the synchronization interface box 9, and the image input terminal of the image processing device 6. The confocal scanner 2 performs rotation synchronization control of the Niipou disc in synchronization with the video signal 101.

  When a video tape deck is used for the image processing apparatus 6, the video tape deck records the video signal 101 input from the image input terminal and the start signal 103 input from the audio input terminal simultaneously on the video tape for a long time. To do. On the video tape, the confocal image that changes in real time and the scanning start timing of the focal position of the objective lens 5 are recorded simultaneously.

  The synchronous interface box 9 extracts either the even-numbered pulse train or the odd-numbered pulse train of the video signal 101 and creates an internal A signal. The arbitrary waveform generator 7 generates a trigger signal 102 which is an H level pulse signal, inputs the trigger signal 102 to the trigger input terminal of the synchronous interface box 9, and uses it for the start timing of scanning of the focal plane.

  The synchronization interface box 9 creates an internal B signal in synchronization with the falling edge of the trigger signal 102. The internal B signal is a pulse signal having a pulse width time of H level of about 35 msec and slightly longer than the video rate time of the video rate camera 1. The synchronous interface box 9 generates a start signal 103 by performing an AND operation on the inverted signal of the internal A signal and the internal B signal, and inputs the start signal 103 to the synchronous input terminals of the image processing device 6 and the arbitrary waveform generator 7.

  The image processing device 6 starts capture for converting and recording the video signal 101 into image data in synchronization with the rising edge of the start signal 103 from the synchronization input terminal. Based on the video signal 101 from the signal input terminal, the synchronization interface box 9 controls rotation synchronization of the Niipou disc by the confocal scanner 2, the start timing of acquisition of the video signal by the image processing device 6, and the objective lens of the optical control system. All the timings for starting the scanning of the focal position are synchronized.

  The arbitrary waveform generator 7 starts scanning the focal position of the objective lens 5 by the optical control system in synchronization with the rise of the start signal 103. The arbitrary waveform generator 7 generates a scanning signal 104 and inputs it to the controller 8. The scanning signal 104 is a sawtooth signal that increases linearly from L level to H level in a certain time. The controller 8 inputs the scanning signal 104 to the actuator 4. The actuator signal 105 is an actual actuator position signal, and after having fully extended, there is an overshoot after returning to the origin at once, and during this time, there is a dead zone.

  The actuator 4 is attached between the objective lens revolver of the microscope 3 and the objective lens 5, and the length in the focal direction of the image changes in proportion to the level of the scanning signal 104 by piezo driving, and the focal position of the objective lens 5 To control. The confocal microscope acquires a slice image of the sample by scanning the focal plane based on the scanning signal 104.

  According to such a configuration, the rotation synchronization control of the Niipou disc, the acquisition timing of the video signal by the image processing device, and the scanning start timing of the focal position of the lens by the optical control system are all synchronized with the video signal, The position accuracy of the confocal image is improved, and variations in individual acquisition times are eliminated when acquiring a plurality of slice images, so that a highly reliable slice image can be obtained.

FIG. 9 is a configuration diagram showing an example of a conventional confocal microscope.
In FIG. 9, the confocal scanner 110 is connected to the port 122 of the microscope 120, and the laser light 111 is condensed into individual light beams by the microlens 117 of the microlens array disk 112 and transmitted through the dichroic mirror 113. , The light passes through individual pinholes 116 of a pinhole array disk (hereinafter referred to as “Nipou disk”) 114 and is focused on the sample 140 on the stage 123 by the objective lens 121 of the microscope 120.

The fluorescent signal emitted from the sample 140 passes through the objective lens 121 again and is collected on each pinhole of the Niipou disk 114. The fluorescence signals that have passed through the individual pinholes are reflected by the dichroic mirror 113 and emitted from the confocal scanner 110 so as to form an image on the image sensor 131 via the relay lens 115. In such an apparatus, the Nipkow disk 114 is rotated at a constant speed by a motor (not shown), and the condensing point on the sample 140 is scanned by the movement of the pinhole 116 by this rotation.

Since the plane on which the pin poles of the Niipou disc 114 are arranged, the plane to be observed of the sample 140, and the light receiving surface of the image sensor 131 are optically conjugate with each other, the optical of the sample 140 is placed on the image sensor 131. A cross-sectional image, that is, a confocal image is formed (see, for example, Patent Document 2).
In addition to the above-described Niipou disc type confocal microscope, there is also a type that acquires a confocal image by scanning a condensing point on a sample using a galvanometer mirror (for example, see Patent Document 3).

JP 2002-72102 A Japanese Patent Laid-Open No. 5-60980 JP-A-5-210051

In image measurement using such a confocal microscope, there is a demand to observe a state change (photoactivation, FRAP (fluorescence recovery after photobleaching), etc.) over time by performing light stimulation on a sample such as a cell. . In photoactivation, for example, a predetermined portion of a cell is irradiated with spot light of a second laser other than the laser beam for image measurement, and the fluorescent color of the portion is changed for marking. The marking is observed to spread over the cells over time.
Further, FRAP (fluorescence fading method) is a method in which the fluorescence of cells expressing a fluorescent protein is partially faded by irradiation with a second laser beam to observe changes in the localization of proteins after fluorescence fading in cells. Is.

However, none of the above-mentioned conventional Niipou disc confocal microscopes have a function of applying a light stimulus to a sample and observing the change.
Further, in a galvanomirror confocal microscope, it takes time to control the galvanometer mirror for two-dimensional scanning, and it is difficult to measure images in real time for high-speed reactions such as light stimulation and fluorescent fading.
Furthermore, it is considered that the light stimulus does not reach or is insufficient for a sample having a three-dimensional structure. As a result, there is a problem that the result of observation of the sample after light stimulation lacks accuracy.

  The present invention is intended to solve the problem of such a conventional confocal microscope, and a function of irradiating a second laser light for light stimulation is added to the Nipo Disc confocal microscope. It is an object to realize a three-dimensional confocal microscope system that can accurately observe a sample after light stimulation three-dimensionally by performing three-dimensional image measurement using the confocal microscope. .

  The present invention is a confocal microscope having the following configuration.

(1) a confocal scanner that acquires a slice image of a sample as a confocal image through a microscope;
A video camera that outputs image data of the confocal image;
An actuator for moving the focal position of the objective lens of the microscope in the optical axis direction;
A control unit that generates a scanning waveform signal for scanning the objective lens in the optical axis direction via the actuator;
An image processing unit that generates a three-dimensional image of the sample based on image data acquired by the video camera;
A three-dimensional confocal microscope system comprising:
Laser light output means connected to the microscope and outputting laser light for stimulating the sample;
A three-dimensional confocal microscope system characterized in that the objective lens is scanned to stimulate the sample in the optical axis direction.

(2) The three-dimensional confocal microscope system according to (1), further comprising variable aperture means for adjusting a spot diameter of a laser beam that gives a stimulus to the sample.

(3) The three-dimensional confocal microscope system according to (1) or (2), further comprising a scanning unit that two-dimensionally scans a laser beam that gives a stimulus to the sample.

(4) The laser beam output means multiplexes and outputs a laser beam for stimulating the sample and a laser beam for indicating a position where the laser beam is irradiated onto the sample. The three-dimensional confocal microscope system according to any one of (1) to (3).

(5) A stimulus is given by scanning in one direction of the objective lens, and then the confocal image is acquired by scanning in another direction, according to any one of (1) to (4) 3D confocal microscope system.

(6) The three-dimensional confocal point according to any one of (1) to (5), wherein a scanning waveform for stimulating the sample and a scanning waveform for observing the sample are each arbitrarily set Microscope system.

(7) The three-dimensional confocal microscope system according to any one of (1) to (6), wherein the three-dimensional confocal microscope system is used for photoactivation or fluorescent fading.

The present invention has the following effects.

According to the first and seventh aspects of the present invention, photoactivation, FRAP, and the like can be performed by providing a nipou disc type confocal microscope with a function of irradiating laser light for light stimulation.
In addition, since fluorescence observation, that is, image measurement, can be performed at high speed (for example, a scanning speed of 1000 frames / second) by using a Niipou disc type confocal scanner, high-speed reactions related to light stimulation and fluorescence fading can be observed in real time. Can do.
Further, by performing three-dimensional image measurement using such a confocal microscope, it is possible to realize a confocal microscope that can accurately observe a sample after light stimulation in three dimensions.

According to the second aspect of the present invention, the variable aperture means is provided, and by changing the irradiation NA (numerical aperture) of the laser beam for photostimulation, the spot diameter of the photostimulation is made variable and the range of photostimulation is increased. You can change the height.

According to the third aspect of the present invention, the light stimulation according to the shape of the sample is made possible by the two-dimensional scan of the laser light for light stimulation.

According to the fourth aspect of the invention, the laser light for light stimulation is irradiated with the laser light for stimulating the light and the laser light for indicating the position where the stimulus is applied, and irradiating the sample. Even in the case of ultraviolet light, since the fluorescence wavelength excited by the position display laser light is visible light, the point of light stimulation can be visually observed.

According to the fifth aspect of the present invention, for example, light stimulation is performed in the ascending direction of the objective lens drive (moving the focal position from the bottom to the top of the sample), and the down direction of the objective lens driving (the focal position is shifted from the top of the sample). The time from the light stimulus to the image measurement can be shortened by performing the image measurement by moving it down.

According to the sixth aspect of the present invention, for example, optical stimulation is performed by linearly scanning up the objective lens drive, and different waveforms are combined such that the objective lens drive down is scanned with a step waveform. Thus, the sample can be observed with a scanning waveform suitable for optical stimulation and image measurement.

  Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing an embodiment of a three-dimensional confocal microscope system according to the present invention. Constituent elements that are the same as in the previous figures are given the same reference numerals, and descriptions thereof are omitted.

  In FIG. 1, the confocal scanner 2a is a nipou disk type similar to that described in the prior art, and has a nipou disk having a large number of pinholes and a microlens array corresponding thereto.

The confocal scanner 2a is attached to a first port 24 (camera port) of a microscope (fluorescence microscope) 3a via an optical path 22 to constitute a confocal microscope. The confocal microscope uses a laser beam to input a fluorescence image of the sample 20 to the confocal scanner 2a via the objective lens 5a, the actuator 4a, and the fluorescence microscope 3a. The confocal scanner 2 a obtains a confocal image of the sample 20 and inputs the confocal image to the camera 1 a via the optical path 23. The camera 1a captures a confocal image and acquires image data. The camera 1a, the confocal scanner 2a, and the fluorescence microscope 3 are installed on a support base 21.

The image measurement laser light source 30 supplies a laser beam having a wavelength λ1 for performing fluorescence excitation for obtaining a confocal image of the sample 20 to the confocal scanner 2a via the fiber 31. The photostimulation laser light source 32 is attached to the second port 25 of the fluorescence microscope 3a via a fiber 33, and supplies a laser beam having a wavelength λ2 for stimulating light. The sample 20 is a cell to be observed and is fixed to the stage 26. The stage 26 is attached to a stage support portion 27.

The synchronization signal controller 10 receives the vertical synchronization signal output from 2CH of the waveform generator 7a, and generates various trigger signals based on the vertical synchronization signal.
When the waveform generator 7a receives the scan waveform generation trigger signal from the synchronization signal controller 10, the waveform generator 7a generates a scan waveform sent from the waveform calculation unit 11 of the personal computer 28 (hereinafter referred to as PC 28) and stored in advance from 1CH. To the actuator driver 12.

The waveform calculation unit 11 calculates a scanning waveform from the scanning period of the objective lens 5a when observing the sample 20 and the scanning distance in the optical axis direction, and outputs it to the waveform generator 7a as scanning waveform data.
The actuator driver 12 generates a drive signal for driving the actuator 4a based on the scanning waveform signal output from the waveform generator 7a.
A portion that generates a scanning waveform signal for scanning the objective lens 5a in the optical axis direction via the actuator 4a is a control unit.

The actuator 4a is attached between the objective lens revolver of the fluorescence microscope 3a and the objective lens 5a, and the length in the focal direction of the image changes in proportion to the level of the scanning signal by piezo driving, and the focal position of the objective lens 5a. To control. That is, the objective lens 5a is scanned in the optical axis direction according to the output waveform of the waveform generator 7a.
Thereby, three-dimensional light stimulation can be performed on the sample, and a slice image in the optical axis direction of the sample 20 is acquired by the camera 1a during image measurement.

  The actuator 4a is provided with a position sensor (not shown) that senses its own position. This position sensor outputs a position signal and feeds it back to the actuator driver 12a, whereby the position of the actuator 4a is controlled.

The image processing unit 6a of the PC 28 receives image data from the camera 1a in synchronization with the start of scanning of the objective lens 5a, and continuously acquires cross-sectional slice images of the sample 20. By synthesizing these slice images, a three-dimensional image of the sample is generated.

The operation of such a configuration will be described with reference to the time chart of each signal shown in FIG.
FIG. 2 is a time chart of each signal handled by the three-dimensional confocal microscope system shown in FIG. The horizontal axis indicates time, and the vertical axis indicates the signal level.

The waveform generator 7a outputs a periodic pulse signal (vertical synchronization signal (a)) in accordance with the frame rate of image capture set by the user. The synchronization signal controller 10 generates various trigger signals based on this periodic pulse signal.

When a vertical synchronization signal (a) having a preset period is sent from the waveform generator 7a to the synchronization signal controller 10, the synchronization signal controller 10 sends the signal to the confocal scanner 2a based on the vertical synchronization signal (a). A synchronization signal for performing rotation synchronization control of the Nipkow disk is sent, and various trigger signals, that is, a scanning waveform generation trigger signal (b) for light stimulation and a scanning waveform generation trigger signal (d) for image measurement are generated.

Further, the synchronization signal controller 10 outputs the vertical synchronization signal (a) as it is to the camera 1a, and the camera 1a captures an image in synchronization with the vertical synchronization signal (a) and stores it in the internal memory. In the stored image data, when the memory is full, the oldest data is overwritten with the latest data.

  When the synchronization signal controller 10 receives the image capture start signal from the PC 28, the scanning waveform generation trigger signal (b) for optical stimulation synchronized with the first vertical synchronization signal after the image capture start signal becomes LOW. ) To the waveform generator 7a.

At the same time, an irradiation ON / OFF signal (c) synchronized with the scanning waveform generation trigger signal (b) is transmitted to the photostimulation laser light source 32, and a built-in shutter (not shown) is opened to provide a photostimulation laser. Turn on the irradiation.
The irradiation time of the laser light for light stimulation is the time from the start to the end of scanning of the focal position for performing light stimulation, and is set in the synchronization signal controller 10 from the PC.
Note that the image capture start signal is a signal input from the PC 28 to the synchronization signal controller 10 by the operator at an arbitrary timing, and the pulse width thereof is twice or more the period of the vertical synchronization signal.

When the waveform generator 7a receives the scanning waveform generation trigger signal (b), the waveform generator 7a generates a scanning waveform signal (f1) based on the scanning waveform data previously sent from the waveform calculation unit 11 in the PC 28, and the actuator driver 12 To drive the actuator 4a. Since the focal position changes according to the signal level of the scanning waveform, the change of the scanning waveform signal (f1) is shown as the change of the focal position.
Thus, the focal plane of the objective lens 5a is continuously changed by driving the actuator 4a, and the sample 20 is subjected to light stimulation three-dimensionally.

When the light stimulation operation is completed, the synchronization signal controller 10 transmits a scanning waveform generation trigger signal (d) for image measurement to the waveform generator 7a and captures an image synchronized with the vertical synchronization signal (a) to the PC 28. Send signal (g).
At the same time, the image measurement laser irradiation ON / OFF signal (e) is transmitted to the confocal scanner 2a, and the built-in shutter (not shown) is opened to turn on the image measurement laser irradiation.

When the waveform generator 7a receives the scanning waveform generation trigger signal (d), the waveform generator 7a generates a scanning waveform signal (f1) based on the scanning waveform data previously sent from the waveform calculation unit 11 in the PC, and the actuator driver 12 To drive the actuator 4a.
The PC 28 receives the image capture signal (g) and causes the camera 1a to output image data in response to a camera control signal synchronized with the image capture signal (g).

As described above, the focal plane of the lens is continuously changed by driving the actuator 4a, and the image data obtained by continuously generating the slice slice image of the sample by the camera 1a in synchronization with the scanning of the actuator 4a is used as the image processing unit of the PC 28. In 6a, a 3D image is synthesized.

The scanning waveform signal (f1) is a step waveform (a waveform that increases stepwise and returns to the original level), and performs fluorescent image measurement at each step (flat portion) in the depth (optical axis) direction of the sample. Therefore, a clear image can be acquired.

A waveform as shown in FIG. 3 may be used as the scanning waveform for scanning the actuator 4a.
FIG. 3 is another time chart of each signal handled in the three-dimensional confocal microscope system shown in FIG. Signals similar to those in FIG. 2 are given the same names and reference numerals, and descriptions thereof are omitted.

In FIG. 3, scanning waveforms (f2), (f3), and (f4) exemplify scanning waveforms output by the waveform generator 7a when receiving the waveform generation trigger signal (d). The scanning waveform data sent from the waveform calculation unit 11 in the PC 28 is converted into an analog waveform. The actuator is driven using any one of these scanning waveforms.

According to the scanning waveform (f2), the optical stimulation is performed at the step of the objective lens driving (moving the focal position from the bottom to the top of the sample) for each step of the step waveform, and the objective lens driving is performed downward (the focal position is changed to the sample position). Move the image from top to bottom) to measure the image. Thereby, the time from optical stimulation to image measurement can be shortened.

According to the scanning waveform (f3), using the isosceles triangular scanning waveform, the optical stimulation is performed in the ascending direction of the objective lens drive, and the image measurement is performed in the descending direction of the objective lens driving, similarly to the scanning waveform (f2). As a result, it is possible to completely perform light stimulation in the depth direction on the sample. Also, in image measurement, the focal position moves in the depth direction of the sample while one frame is imaged, so that an image obtained by averaging the confocal images between them (averaged light intensity) can be captured. The image information in the depth direction of the sample is acquired without fail.

Further, as shown in the scanning waveform (f4), the objective lens drive may be linearly scanned to perform light stimulation, and the objective lens drive descend may be scanned with a step waveform. By combining such different waveforms, the sample can be observed with a scanning waveform suitable for optical stimulation and image measurement.

Here, the configuration of a confocal microscope applied to such a three-dimensional confocal microscope system will be described with reference to the drawings. In the following embodiments, a confocal microscope having a confocal scanner mounted on the microscope is shown, but the confocal microscope is equivalent to that shown in FIG.
FIG. 4 is a block diagram showing an embodiment of a confocal microscope applied to the present invention. Constituent elements similar to those in the previous drawings are denoted by the same reference numerals, and description thereof is omitted.
In FIG. 4, a confocal scanner 110 is attached to the first port 41 of the microscope 40 to configure a confocal microscope that irradiates a sample 140 with a laser beam 111 (first laser beam) having a wavelength λ1. The laser beam 111 that has entered the microscope 40 passes through the dichroic mirror 43 and is focused on the sample 140 such as a cell on the stage 46 by the objective lens 44. The sample 140 is fluorescent by the irradiation of the laser beam 111. The fluorescence signal emitted from the sample 140 passes through the objective lens 44 again, passes through the dichroic mirror 43, and forms an image on the image sensor 131 through the confocal scanner 110 as in the conventional example.

Laser light output means 50 is attached to the second port 42 of the microscope 40. The laser light output means 50 includes a laser light source 51 and a collimator lens 52. The laser light source 51 emits a second laser beam 53 having a wavelength of λ2, and the collimator lens 52 converts the second laser beam 53 into parallel light and enters the microscope 40 from the second port 42. The second laser beam 53 that has entered the microscope 40 is reflected by the dichroic mirror 43, and the objective lens 44 forms an image of the light beam spot of the laser light for light stimulation having the wavelength λ 2 on the sample 140.
In this case, the relationship between λ1 and λ2 is λ2 <λ1.

By providing the Nipo Disc confocal microscope with the function of irradiating the second laser beam for light stimulation, it becomes possible to perform photoactivation application, FRAP, and the like. In addition, since fluorescence observation, that is, image measurement, can be performed at high speed (for example, a scanning speed of 1000 frames / second) by using a Niipou disc type confocal scanner, high-speed reactions related to light stimulation and fluorescence fading can be observed in real time. Can do.

Here, in the configuration shown in FIG. 4, when the second laser beam 53 is not visible light such as ultraviolet light, the irradiation point of the second laser cannot be visually observed. In order to solve this problem, as shown in FIG. 5, the laser spot of visible light is superimposed on the beam spot of the second laser light.

FIG. 5 is a block diagram showing a second embodiment of the confocal microscope applied to the present invention. Constituent elements similar to those in FIG. 4 are given the same reference numerals, and descriptions thereof are omitted.
In FIG. 5, a confocal scanner 110 is attached to the first port 41 of the microscope 40 to constitute a confocal microscope that irradiates a laser beam 111 having a wavelength λ1. The laser beam 111 entering the microscope 40 passes through the dichroic mirror 43 and is focused on the sample 140 on the stage 46 by the objective lens 44. The sample 140 is fluorescent by the irradiation of the laser beam 111. The fluorescence signal emitted from the sample 140 passes through the objective lens 44 again, passes through the dichroic mirror 43, and forms an image on the image sensor 131 through the confocal scanner 110 as in the conventional example.

Laser light output means 60 is attached to the second port 42 of the microscope 40. The laser light output means 60 includes laser light sources 61 and 65, collimator lenses 62 and 66, a dichroic mirror 64, a total reflection mirror 67, and a variable aperture means 68.

The laser light source 61 emits a second laser beam 63 having a wavelength λ2 indicated by a solid line. The collimator lens 62 makes the second laser light 63 parallel light. The dichroic mirror 64 transmits the second laser light 63 that has become parallel light due to its spectral characteristics. The variable aperture means 68 varies the beam diameter of the second laser light 63 that has passed through the dichroic mirror 64. The second laser light 63 that has passed through the variable aperture means 68 enters the microscope 40 from the second port 42 of the microscope 40. The second laser light 63 that has entered the microscope 40 is reflected by the dichroic mirror 43, and the objective lens 44 forms an image of a light beam spot of the laser light for light stimulation having the wavelength λ 2 on the sample 140.

The laser light source 65 emits a third laser beam 69 having a wavelength λ3 indicated by a broken line. The collimator lens 66 makes the third laser light 69 parallel light. The total reflection mirror 67 reflects the third laser light 69 that has become parallel light and applies it to the dichroic mirror 64. The dichroic mirror 64 reflects the third laser beam 69 due to its spectral characteristics, and enters the microscope 40 from the second port 42 via the variable aperture means 68. The third laser light 69 entering the microscope 40 is reflected by the dichroic mirror 43 and forms an image of a light beam spot having a wavelength λ3 on the sample 140 by the objective lens 44.

As a result, the second fluorescent signal 45 is generated. The second fluorescent signal 45 passes through the objective lens 44 again, passes through the dichroic mirror 43, and forms an image on the image sensor 131 through the confocal scanner 110 in the same manner as the fluorescent signal generated by the laser beam 111. The relationship between λ1, λ2, and λ3 is λ2 <λ1 <λ3. The second fluorescent signal 45 is visible light and has a wavelength longer than λ3.

By combining the light of λ2 and λ3 and irradiating the sample 140, the fluorescence wavelength excited by the third laser light (λ3) is visible even if the second laser light (λ2) is ultraviolet light. Then, the point where light stimulation is performed becomes visible.
Further, by providing the variable aperture means 68 and changing the irradiation NA (numerical aperture) of the second and third laser beams, the spot diameter of the light stimulation can be made variable, and the size of the light stimulation range can be changed. I have to.

FIG. 6 is a block diagram showing a third embodiment of the confocal microscope applied to the present invention. Constituent elements similar to those in the previous drawings are denoted by the same reference numerals, and description thereof is omitted.
In FIG. 6, the configurations of the confocal scanner 110 and the microscope 1 are the same as those shown in FIG.
Laser light output means 70 is attached to the second port 42 of the microscope 40. The laser light output means 70 includes laser light sources 61 and 65, collimator lenses 62 and 66, a dichroic mirror 64, a total reflection mirror 67, and a scanning means 71.

The configurations and operations of the laser light sources 61 and 65, the collimator lenses 62 and 66, the dichroic mirror 64, and the total reflection mirror 67 are the same as those in the second embodiment shown in FIG. 5, and the scanning means 71 is added to this configuration.

The scanning means 71 constitutes a mirror scan type scanning system. Although not shown, the laser beam is scanned two-dimensionally using, for example, a galvanometer mirror. The galvanometer mirror is a mechanism that can be rotated in the vertical and horizontal directions by a DC motor. A two-dimensional arbitrary position can be irradiated with a laser spot by rotating a galvanometer mirror by controlling a DC motor according to a signal from a control unit.

The second and third laser beams 63 and 69 output from the scanning unit 71 enter the microscope 40, are reflected by the dichroic mirror 43, are collected by the objective lens 44, and irradiate the sample 140 with spot light. By two-dimensional scanning of the second and third laser beams 63 and 69 of the scanning unit 71, light stimulation in accordance with the shape of the sample 140 is made possible. Similarly to the second embodiment, even if the second laser beam 63 that provides optical stimulation is ultraviolet light, the third laser beam 69 can visually recognize the portion that provides stimulation.

When observation of light stimulation is performed on a sample in a two-dimensional plane, light stimulation may not reach or be insufficient in the depth direction of a sample having a three-dimensional structure. Conceivable. As a result, the result of sample observation after light stimulation lacks accuracy.
In addition, when a part of cells as a sample is marked by light stimulation, it is impossible to accurately observe how the marking spreads in the depth direction as well as the planar direction.
In order to solve such a problem, three-dimensional image measurement is performed by using a Niipou disc type confocal microscope having a light stimulation function as described above, and thus the observation of the sample after the light stimulation is performed three-dimensionally. Can be performed accurately.

  The present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.

It is a block diagram which shows one Example of the three-dimensional confocal microscope system which concerns on this invention. It is a time chart of each signal handled with the three-dimensional confocal microscope system which concerns on this invention. It is another time chart of each signal handled with the three-dimensional confocal microscope system which concerns on this invention. It is a block diagram which shows one Example of the confocal microscope applied to this invention. It is a block diagram which shows the 2nd Example of the confocal microscope applied to this invention. It is a block diagram which shows the 3rd Example of the confocal microscope applied to this invention. It is a block diagram which shows an example of the conventional confocal microscope system. It is a time chart of the various signals which the confocal microscope system of FIG. 7 handles. It is a block diagram which shows an example of the conventional confocal microscope.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1a Camera 2a Confocal scanner 3a Fluorescence microscope 4a Actuator 5a Objective lens
6a Image processing unit 7a Waveform generator 10 Synchronization signal controller 11 Waveform calculation unit 12 Actuator driver 20 Sample 21 Support base 22 Optical path 23 Optical path 24 First port 25 Second port 26 Stage 27 Stage support unit 28 PC
DESCRIPTION OF SYMBOLS 30 Image measurement laser light source 31 Fiber 32 Optical stimulation laser light source 33 Fiber 40 Microscope 41 1st port 42 2nd port 43 Dichroic mirror 44 Objective lens 46 Stage 50 Laser light output means 51 Laser light source 52 Collimator lens 53 2nd laser Light 60 Laser light output means 61 Laser light source 62 Collimator lens 63 Second laser light 64 Dichroic mirror 65 Laser light source 66 Collimator lens 67 Total reflection mirror 68 Variable aperture means 69 Third laser light 70 Laser light output means 71 Scanning means 110 Confocal scanner

Claims (7)

A confocal scanner that acquires a slice image of a sample as a confocal image through a microscope;
A video camera that outputs image data of the confocal image;
An actuator for moving the focal position of the objective lens of the microscope in the optical axis direction;
A control unit that generates a scanning waveform signal for scanning the objective lens in the optical axis direction via the actuator;
An image processing unit that generates a three-dimensional image of the sample based on image data acquired by the video camera;
A three-dimensional confocal microscope system comprising:
Laser light output means connected to the microscope and outputting laser light for stimulating the sample;
A three-dimensional confocal microscope system characterized in that the objective lens is scanned to stimulate the sample in the optical axis direction.   The three-dimensional confocal microscope system according to claim 1, further comprising variable aperture means for adjusting a spot diameter of a laser beam that gives a stimulus to the sample.   The three-dimensional confocal microscope system according to claim 1, further comprising a scanning unit configured to two-dimensionally scan a laser beam that gives a stimulus to the sample. 2. The laser beam output means multiplexes and outputs a laser beam for stimulating the sample and a laser beam for indicating a position where the laser beam is irradiated on the sample. The three-dimensional confocal microscope system according to claim 3.   5. The three-dimensional image according to claim 1, wherein a stimulus is given by scanning in one direction of the objective lens, and subsequently the confocal image is acquired by scanning in the other direction. Focus microscope system.   6. The three-dimensional confocal microscope system according to claim 1, wherein a scanning waveform for stimulating the sample and a scanning waveform for observing the sample are arbitrarily set. The three-dimensional confocal microscope system according to any one of claims 1 to 6, wherein the three-dimensional confocal microscope system is used for photoactivation or fluorescent fading.

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