JP2003315681A - Microscope apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope apparatus which can scan arbitrary positions respectively independently a first scanning optical system and a second scanning optical system at an arbitrary time. <P>SOLUTION: The microscope apparatus is provided with the first scanning optical system A for obtaining the scanning image of a specimen and the second scanning optical system B for developing a specific phenomenon in the specific area of the specimen, means 30 for performing laser scanning independently with each of the first scanning optical system and the second scanning optical system and operation recording means 23 for recording the operation of the laser scanning. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope, and more particularly to a microscope device equipped with a laser scanning means for generating a peculiar phenomenon on a specimen.

[0002]

2. Description of the Related Art Fluorescence observation is known as one of observations of biological specimens with a microscope. This fluorescence observation is to prepare a sample in which an ion-sensitive fluorescent indicator is introduced into a biological sample, irradiate the sample with excitation light, and measure the fluorescence emitted from the sample. In this case, there is a certain correlation between the brightness of fluorescence and the concentration of ions such as calcium ions. Therefore, the concentration of calcium ions can be indirectly measured by fluorescence observation. Utilizing this fact, it is possible to measure the change with time of the calcium ion concentration distribution in the organism.

Furthermore, recently, a caged reagent capable of causing an ionic substance to appear at a specific site in a sample at a specific time has been used as an application for observing a biological sample. The caged reagent is one in which an ionic substance such as calcium ion is included in a caged group serving as a shell. When the caged reagent is irradiated with ultraviolet rays after the caged reagent is drawn into the sample, the caged group is cleaved. Thereby, an ionic substance can be made to appear in a desired specific site at a specific time, and changes in the biological specimen before and after that can be observed. Thus, for example, a phenomenon in which a caged group is cleaved to cause an ionic substance to appear at a desired specific site is referred to as a “singular phenomenon”, but the unique phenomenon is not limited to this, but an action (for example, caged Release, fluorescence bleaching, etc.) and all the phenomena generated at a desired specific site.

Further, there are applications called FLIP and FRAP as applications for observing biological specimens.
In these methods, a part of a biological specimen is stimulated or damaged by light, and the change with time of the specimen before and after that is measured.

All of the applications of the microscope as described above control the position and time of light irradiation and require complicated light irradiation operations.

As a microscope applied to the above-mentioned applications, a first scanning optical system (laser light source, scanner, objective lens) for observing a sample and a second scanning optical system for stimulating a sample (caged cleavage) are used. A laser microscope provided with two optical systems separately has been proposed (JP-A-10-20).
6742). However, this laser microscope is not provided with means for recording the temporal relationship between the first scanning optical system and the second scanning optical system. Therefore, it is difficult to define the observation time that is essential for applications using caged cleavage.

In order to solve the above problems, a scanning laser microscope has been proposed which synchronizes the light irradiation timings of the first scanning optical system and the second scanning optical system (Japanese Patent Laid-Open No. 2000-275529). (See Japanese Patent Publication). In this way, the observation time can be accurately determined by synchronizing the light irradiation timings of the first scanning optical system and the second scanning optical system.

However, in this scanning laser microscope, although the temporal relationship can be guaranteed by synchronizing the first scanning optical system and the second scanning optical system, on the other hand, the second scanning optical system is It must operate in synchronism with the first scanning optics. Therefore, the second scanning optical system cannot be operated or stopped at any time, and measurement based on the state of the sample cannot be performed. As a result, there is a risk of narrowing the application range of the microscope observation.

Further, an optical path for image acquisition and an optical path for stimulating the sample are integrated in front of the scanner, and a high-speed shutter which can be electrically controlled is arranged at the emission port of the laser for stimulation. Type microscopes have also been proposed (Japanese Patent Application Laid-Open No. H9-9119).
-329750). However, since this laser scanning microscope has only one scanner, it cannot irradiate a laser for sample stimulation at another scanning speed during sample image acquisition.

[0010]

As described above, none of the conventional laser scanning microscopes can irradiate a laser beam to an arbitrary position for an arbitrary time.

It is an object of the present invention to provide a microscope apparatus capable of independently scanning a first scanning optical system and a second scanning optical system at arbitrary locations on a sample at arbitrary times. Another object of the present invention is to provide a microscope device that can guarantee the scanning time and place.

[0012]

The present invention has taken the following means in order to solve the above problems. A microscope apparatus according to the present invention includes a first scanning optical system for obtaining a scanned image of a specimen, a second scanning optical system for expressing a peculiar phenomenon in a specific portion of the specimen, and the first scanning. Each of the optical system and the second scanning optical system is provided with means for independently controlling the operation of laser scanning and operation recording means for recording the operation of the laser scanning.
In the above microscope apparatus, it is preferable that the first scanning optical system and the second scanning optical system each have a shutter.

Another microscope apparatus according to the present invention has a lamp as a light source, a charge storage element array as a light receiving element, an optical system for obtaining an image of a sample, and a peculiar phenomenon in a specific part of the sample. Laser scanning optical system for
It is characterized by comprising a time for acquiring an image obtained by the charge storage device array and a unit for recording the operation of the laser scanning optical system. In the microscope apparatus described above, it is preferable that the optical system for obtaining the image of the sample and the laser scanning optical system each have a shutter.

[0014]

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

(First Embodiment) FIG. 1 shows a first embodiment of the present invention.
It is a block diagram of an optical system of the microscope apparatus according to the embodiment of.

The microscope apparatus according to the first embodiment of the present invention includes a first scanning optical system A and a second scanning optical system B. The first scanning optical system A is an observation scanning optical system that scans the laser light from the first laser light source 1 on the focal plane of the sample 19. The second scanning optical system B is a scanning optical system for irradiating a laser beam output from the second laser light source 11 to an arbitrary position of the sample to cleave the caged reagent.

The first scanning optical system A comprises a first laser light source 1, a dichroic mirror 2, a first laser shutter 3 (acousto-optical element capable of high speed operation, etc.), and a first scanning optical unit 4. And a relay lens 5 and a mirror 6.

In the first scanning optical system A, a detection optical system C is arranged on the branched optical path of the dichroic mirror 2. The detection optical system C has a filter 7, a lens 8, a confocal pinhole 9, and a photoelectric conversion element 10.

The second scanning optical system B includes a second laser light source 11 for cleaving the caged reagent, a second laser shutter 12 (acousto-optical element capable of high speed operation, etc.),
A second scanning optical unit 13, a relay lens 14,
It has a dichroic mirror 15.

The optical axis of the first scanning optical system A and the optical axis of the second scanning optical system B are combined (matched) by the dichroic mirror 15 and guided to the imaging lens 16 and the objective lens 17. Get burned. Further, the focal positions of the relay lens 5 and the relay lens 14 are arranged so as to coincide with the focal position of the imaging lens 16. The dichroic mirror 15 transmits light having a wavelength longer than the wavelength of the laser light from the first scanning optical system A, and at the same time, the second scanning optical system B.
It has a characteristic of reflecting the wavelength of the laser beam from.

The sample 19 into which the caged reagent has been introduced is placed on the stage 18.

The first and second laser shutters 3 and 12
The first and second optical scanning units 4, 13 and the photoelectric conversion element 10 are connected to the optical system control means 21.
A CRT display 22 and an operation recording means 23 are connected to the optical system control means 21.

The operation of the microscope apparatus configured as described above will be described. The laser light from the second laser light source 11 is
The passage or blocking is controlled by opening and closing the optical system control means 21. The laser light passes when the first laser shutter 3 is in the open state, is guided to the first scanning optical unit 4 which is scan-controlled by the optical system control means 21, and is deflected and scanned in an arbitrary direction. . The laser light is further focused on the cross section 20 of the sample 19 via the relay lens 5, the mirror 6, the dichroic mirror 15, the imaging lens 16, and the objective lens 17, and the cross section 20 is two-dimensionally scanned.

A fluorescence indicator excited by the wavelength of the first laser light source 1 is introduced into the sample 19, and the cross section 2
When the laser beam scans over 0, the fluorescent indicator is excited to generate fluorescence. The fluorescence that has entered the objective lens 17 travels in the opposite direction along the same optical path as the above laser light. That is, the fluorescence passes through the objective lens 17, the imaging lens 16, and the dichroic mirror 15, and is guided to the dichroic mirror 2 via the mirror 6, the relay lens 5, and the first scanning optical unit 4. The dichroic mirror 2 is the first
Since it has a characteristic of reflecting light having a wavelength longer than the wavelength of the laser light from the laser light source 1, the fluorescence is reflected by the dichroic mirror 2 and introduced into the detection optical system C.

In the detection optical system C, the fluorescence is filtered by the filter 7
Thus, light of a specific wavelength is selectively transmitted, and only the fluorescence from the cross section 20 enters the photoelectric conversion element 10 through the lens 8 and the confocal pinhole 9.

The output signal from the photoelectric conversion element 10 is guided to the optical system control throw 21 and converted into a digital signal in synchronization with scanning control, and an image corresponding to the scanning position is displayed on the screen of the CRT display 22. To be done. The image displayed on the CRT display screen 22 shows a fluorescent image (two-dimensional distribution of fluorescent brightness) at the cross section 20. The two-dimensional distribution of the fluorescence brightness shows the distribution of the desired ion concentration in the cross section 20.

On the other hand, the laser light from the second laser light source 11 passes through the second laser shutter 12 when the second laser shutter 12 whose opening and closing is controlled by the optical system control means 21 is in the open state, 2 scanning optical system B, relay lens 1
4, combined with the optical axis of the first scanning optical system A via the dichroic mirror 15. Similar to the first scanning optical system A, this laser light passes through the imaging lens 16 and the objective lens 17, and is irradiated onto the cross section 20 of the sample 19. At this time, by controlling the second scanning optical system B by the optical system control means 21, it is possible to select an arbitrary position in the cross section 20 that does not depend on the scanning position of the first scanning optical system A as the irradiation position. it can.

The shutter 3 of the first scanning optical system A and the shutter 12 of the first scanning optical system A are, for example, acousto-optical elements, and the opening / closing speed of these shutters is Since it can be performed at a speed sufficiently higher than the scanning speed per pixel, the laser light can be applied to only a part of the scanning range by opening the shutter only when scanning only a part of the scanning range. it can.

When the laser light from the second laser light source 11 is irradiated onto the specimen 19 into which the caged reagent has been introduced as described above, the caged group of the caged reagent at the irradiated portion is cleaved and the inside thereof is cleaved. The contained substance is released. Then, the change in the ion concentration distribution in the sample 19 due to this emission can be measured by the image obtained by the first scanning optical system A.

Next, an electrical control system centering on the optical system control means 21 will be described. FIG. 2 is a block diagram showing the internal structure of the optical system control means 21 in the first embodiment. The CPU 30 controls the entire system including the control of the optical system. First and second scanning waveform generation circuits 32 and 33
Respectively generate control signals for the first and second optical scanning units 4 and 13. The first and second shutter opening / closing circuits 34 and 35 are provided for the first and second laser shutters 3,
A signal for operating 12 is generated. The first and second optical scanning units 4 and 13 and the first and second laser shutters 3 and 12 include first and second scanning waveform generation circuits 32 and 33.
It is controlled by the signal generated in. The first and second scanning waveform generation circuits 32 and 33 and the first and second shutter opening / closing circuits 34 and 35 can all be controlled independently. In addition, the first and second scanning waveform generation circuits 32 and 33
And the control signals to the first and second shutter opening / closing circuits 34 and 35 are simultaneously sent from the CPU 30 to the operation recording means 23.

The operation recording means 23 comprises a storage device. The operation recording means 23 records information relating to the contents of the instruction from the CPU 30 to each circuit. Further, the image processing circuit 31 samples and stores the luminance information sent from the photoelectric conversion element 10 in synchronization with the sampling clock generated by the first scanning waveform generation circuit 32. The frequency information stored in the image processing circuit 31 is stored in the CRT display 2
2 is output. As a result, the image obtained by scanning the sample 19 can be output to the CRT display 22. Further, the information recorded in the motion recording means 23 and the image are CR.
By displaying on the T display 22, the sample 19
Can be observed over time.

As described above, according to the first embodiment of the present invention, the laser irradiation of the first scanning optical system A and the second scanning optical system B can be performed independently. So, for example,
The caged reagent at an arbitrary position on the sample can be cleaved at an arbitrary time by the second scanning optical system B without being influenced by the first scanning optical system A.

Further, since the operation recording means 23 records the operation of the first scanning optical system A and the second scanning optical system B with the passage of time,
An image observed by the first scanning optical system A and a caged cleavage by the second scanning optical system B can be temporally corresponded.

Further, as an application of the microscope observation, it is possible to adopt a configuration in which the laser light of the second scanning optical system B is cut off according to the brightness value of the image. The flow of operation in such a case will be described with reference to FIGS. 3 and 4.
FIG. 3 is a flowchart for controlling the second scanning optical system B in accordance with the brightness value of the image, and FIG. 4 is a diagram showing changes in the brightness value of the image over time.

First, the scanning by the first scanning optical system A is started (step A1). Then, the end brightness value for canceling the cage is set (input) (step A).
2). This end brightness value may be set in advance or may be appropriately input by the observer. Next, when scanning by the second scanning optical system B is started (step A
3), as shown in FIG. 4, by cleavage of the caged reagent,
The brightness value of the image gradually increases. Then, until the image brightness value reaches the cage removal end brightness value (step A
4) Continue scanning by the second scanning optical system. And
If the brightness value of the image has reached the cage removal end brightness value in step A4, the shutter of the second scanning optical system B is closed (step A5), and this time is
Record as the caged release end time (step A)
6). The cage release end time in step A6 may be an elapsed time from the start time of scanning by the first scanning optical system A or the second scanning optical system B, not the actual time. Then, the scanning by the second scanning optical system B is stopped (step A7).

As described above, after the image brightness is monitored and the cage is cleaved, when the brightness value of the sample reaches the brightness set by the application software, the laser of the second scanning optical system B is automatically operated. Is blocked by the shutter 12. At this time, the time when the shutter 12 shuts off the laser of the second scanning optical system B and the place where the shutter 12 was scanning at that time are recorded in the operation recording means 23. The first embodiment can be applied to such an application that requires a complicated operation of the optical system, and these operations can be automatically performed.

(Second Embodiment) Since the configuration of the microscope apparatus according to the second embodiment is the same as that of the first embodiment,
Illustration and description are omitted. In the second embodiment, the sample 1
It is assumed that the caged reagent is not drawn into the sample 9.

When the fluorescent sample 19 is irradiated with laser light, the sample 1
9 takes damage. Therefore, the portion of the fluorescent sample 19 to which the laser beam is applied has a low ion concentration, and the brightness is weak. However, when the laser light is blocked, the fluorescent brightness gradually recovers due to the diffusion of the fluorescent dye.

By using the second scanning optical system B to damage the sample 19, the above-described change with time of the sample 19 can be observed.

The operation flow in such a case will be described with reference to FIGS. FIG. 5 is a flowchart for controlling the second scanning optical system B according to the brightness value of the image, and FIG. 6 is a diagram showing changes in the brightness value of the image over time. In FIG. 5, the same parts as those in FIG. 3 are designated by the same reference numerals.

First, the scanning by the first scanning optical system A is started (step A1). Then, a threshold value for ending the scanning of the second scanning optical system is set (input) (step B1). This threshold value may be set in advance or may be appropriately input by the observer.
Next, when the scanning by the second scanning optical system B is started (step A3), as shown in FIG. 6, the sample 19 is damaged, so that the luminance value of the image gradually decreases. Then, the scanning by the second scanning optical system is continued until the image brightness value reaches the threshold value (fading) (step B2). Then, in step B2, if the brightness value of the image has reached the threshold value, the shutter of the second scanning optical system B is closed (step A5), and this time is irradiated by the second scanning optical system. It is recorded as the end time of (step A6). The time and time in step A6 may be the elapsed time from the start time of scanning by the first scanning optical system A or the second scanning optical system B, not the actual time. Then, the scanning by the second scanning optical system B is stopped (step A7).

As described above, by utilizing the fact that the image brightness is monitored and the second scanning optical system B is operated and stopped at an arbitrary time, for example, the laser of the second scanning optical system B as described above is used. The sample 19 is damaged by being exposed to light and the brightness is weakened, but when the brightness value is reduced to the threshold value, the laser light of the second scanning optical system B is automatically blocked by the shutter 12. At this time, the time (time) when the shutter 12 of the second scanning optical system B shuts off the laser light is recorded in the operation recording means 23, so that the image and the data can be correlated after the measurement.

As described above, the second embodiment can also be applied to an application requiring a complicated optical system operation, and these operations can be automatically performed.

(Third Embodiment) FIG. 7 is a block diagram of an optical system of a microscope apparatus according to the third embodiment. Figure 7
In the above, since the configuration other than the first scanning optical system A and the detection optical system C is the same as that of the first embodiment,
The same reference numerals are given and detailed description is omitted. In the third embodiment, the first laser light source 1 in the second embodiment is used.
Instead, a lamp 24 is provided.

That is, the first scanning optical system A includes the lamp 24 as the excitation light source and the high-speed shutter 2 of the lamp 24.
9, a collimating lens 25, a dichroic mirror 6 ′ that reflects excitation light and transmits fluorescence from the biological specimen 19, and laser light from the second scanning optical system B to reflect fluorescence from the biological specimen 19. Transparent dichroic mirror 1
5, an imaging lens 16, an objective lens 17, a camera lens 27, and a charge storage type photoelectric conversion element array (for example, CCD) 28.

In the above structure, the brightness information acquired by the CCD 28 of the first scanning optical system A is the optical system control means 2.
Sent to 1 '. At the same time, the time information is sent to the operation recording means 23.

Optical system control means 2 in the third embodiment
The internal structure of 1'is shown in FIG. 8, the same parts as those in FIG. 2 are designated by the same reference numerals, and detailed description thereof will be omitted. In the third embodiment, since the sample is excited by the lamp 24, the first scanning waveform generating circuit 32 and the first optical scanning unit 4 in FIG. 2 are omitted in FIG. Therefore, the control signal from the CPU 30 is output to the image processing circuit 31.

Also in the third embodiment, the second scanning waveform generating circuit 33 and the first and second shutter opening / closing circuits 34 and 35 can be independently controlled as in the first embodiment. In addition, the first and second scanning waveform generation circuits 32 and 33
Also, control signals to the first and second shutter opening / closing circuits 34 and 35 are simultaneously sent from the CPU 30 to the operation recording means 23, and the operation recording means 23 records when and what operation was performed.

As described above, according to the third embodiment,
Since the image is captured by the CCD 28, the number of frames can be increased and the temporal resolution can be improved in observing changes over time such as caged cleavage. Further, since the first scanning optical system can observe a wide and deep region of the specimen, the entire specimen (including the depth direction of the specimen) can be observed when the caged cleavage is performed.

In addition, C is added to the existing laser scanning microscope device.
Since it is possible to provide two optical systems only by adding the CD 28, it is advantageous in terms of cost and an application that requires two scanning optical systems (for example, in the first and second embodiments). Application as shown)
It is possible to build a microscope system compatible with.

The following inventions can be extracted from the above embodiments. The present invention is not limited to the above embodiments. Of course, various modifications can be made without departing from the scope of the invention.

The microscope apparatus according to the present invention comprises a first scanning optical system for obtaining a scanned image of a specimen, a second scanning optical system for expressing a peculiar phenomenon at a specific portion of the specimen, and the first scanning optical system. Means for independently controlling the laser scanning operation for each of the first scanning optical system and the second scanning optical system; and operation recording means for recording the laser scanning operation.
It is characterized by including. In the above microscope apparatus, it is preferable that the first scanning optical system and the second scanning optical system each have a shutter.

Another microscope apparatus according to the present invention has a lamp as a light source and a charge storage type element array as a light receiving element, and an optical system for obtaining an image of a sample and a peculiar phenomenon are generated in a specific part of the sample. Laser scanning optical system for
It is characterized by comprising a time for acquiring an image obtained by the charge storage device array and a unit for recording the operation of the laser scanning optical system. In the microscope apparatus described above, it is preferable that the optical system for obtaining the image of the sample and the laser scanning optical system each have a shutter.

[0054]

According to one embodiment of the present invention, the first optical system and the second optical system can independently scan arbitrary locations on a sample at arbitrary times. Also, the time and place of scanning can be guaranteed.

[Brief description of drawings]

FIG. 1 is a block diagram of an optical system of a microscope apparatus according to a first embodiment of the present invention.

FIG. 2 is a block diagram showing an internal structure of an optical system control means in the first embodiment.

FIG. 3 is a flowchart for controlling a second scanning optical system according to a brightness value of an image.

FIG. 4 is a diagram showing a change in luminance value of an image with the passage of time.

FIG. 5 is a flowchart for controlling the second scanning optical system according to the brightness value of an image.

FIG. 6 is a diagram showing a change in luminance value of an image with the passage of time.

FIG. 7 is a block diagram of an optical system of a microscope apparatus according to a third embodiment.

FIG. 8 is a block diagram showing the internal structure of an optical system control means according to a third embodiment.

[Explanation of symbols]

A ... First scanning optical system B: second scanning optical system C ... Detection optical system 1 ... first laser light source 2 ... Dichroic mirror 3 ... First laser shutter 4 ... First scanning optical unit 5 ... Relay lens 6 ... Mirror 6 '... dichroic mirror 7 ... Filter 8 ... Lens 9 ... Confocal pinhole 10 ... Photoelectric conversion element 11 ... Second laser light source 12 ... Second laser shutter 13 ... Second scanning optical unit 14 ... Relay lens 15 ... Dichroic mirror 16 ... Imaging lens 17 ... Objective lens 18 ... Stage 19 ... Specimen 20 ... Section 21, 21 '... Optical system control means 22 ... CRT display 23 ... Action recording means 24 ... Lamp 25 ... Collimating lens 27 ... Camera lens 28 ... Charge storage type photoelectric conversion element array (CCD) 29 ... High-speed shutter 30 ... CPU 31 ... Image processing circuit 32 ... First scanning waveform generating circuit 33 ... Second scanning waveform generating circuit 34, 35 ... First and second shutter opening / closing circuits

Claims (4)

[Claims] 1. A first scanning optical system for obtaining a scanned image of a specimen, a second scanning optical system for expressing a peculiar phenomenon in a specific portion of the specimen, and the first scanning optical system. A microscope apparatus comprising: a unit that independently controls a laser scanning operation for each of the second scanning optical systems; and an operation recording unit that records the laser scanning operation. 2. The microscope apparatus according to claim 1,
A microscope apparatus, wherein each of the first scanning optical system and the second scanning optical system has a shutter. 3. An optical system having a lamp as a light source and a charge storage element array as a light receiving element, and an optical system for obtaining an image of a sample, and a laser scanning optical system for producing a peculiar phenomenon at a specific portion of the sample. A microscope apparatus comprising: a time when an image obtained by the charge storage element array is acquired; and a unit that records the operation of the laser scanning optical system. 4. The microscope apparatus according to claim 3,
A microscope apparatus, wherein each of an optical system for obtaining an image of the sample and a laser scanning optical system has a shutter.