JP4468642B2 - Confocal laser scanning microscope apparatus and sample information recording method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention belongs to a technical field related to a confocal laser scanning microscope apparatus.
[0002]
[Prior art]
Conventionally, as a scanning microscope apparatus, a laser beam from a light source is narrowed down to a minute spot light by an objective lens, and an observation sample is scanned with the spot light, and light from the observation sample, for example, transmitted light, reflected light, or There is known a laser scanning microscope apparatus that converts fluorescence and the like into an electrical signal by a photoelectric converter such as a photomultiplier and displays an observation image on an image monitor.
[0003]
On the other hand, recently, as a research technique in the fields of neuroscience and cell biology, a method of quantifying intracellular free ion concentration using an ion-sensitive indicator is rapidly spreading. For example, in the measurement of intracellular calcium ion concentration, fluorescence measurement using a fluorescent reagent fura-2 which is a calcium ion sensitive indicator is performed on a cell which is a sample. In this fluorescence measurement, a specific region of a cell is excited with two different wavelengths (340 nm, 380 nm), a stimulating substance (Bradykinin) is administered, and then quantified by determining the ratio of the respective fluorescence intensities. Measurements with a fluorometer, observation with a microscope, and the like are performed.
[0004]
Patent Document 1 discloses a fluorescence microscope apparatus that measures the calcium ion concentration using the aforementioned fluorescent reagent fura-2. In this fluorescence microscope apparatus, by arbitrarily switching a plurality of interference filters having different transmission wavelengths, the sample is irradiated with only light having a wavelength transmitted by the switched filter out of the light source. A sample image is acquired by imaging and processing the light of the light, and using a plurality of lights having different wavelengths as excitation light, such as two-wavelength excitation and one-wavelength fluorescence measurement using the fluorescence reagent fura-2 It can be measured.
[0005]
[Patent Document 1]
JP-A-2-28542 [0006]
[Problems to be solved by the invention]
However, Patent Document 1 does not disclose anything about the configuration in the case of performing the measurement using a confocal laser scanning microscope apparatus, and a normal optical microscope apparatus that simply acquires a two-dimensional image optically. Only the configuration when using is disclosed.
[0007]
Conventionally, any configuration is disclosed in which a confocal laser scanning microscope apparatus is used to perform measurement using a plurality of lights having different wavelengths as excitation light, such as the aforementioned two-wavelength excitation single-wavelength fluorescence measurement. Was not.
In view of the above circumstances, an object of the present invention is to provide a confocal laser scanning microscope apparatus and a sample information recording method applicable to measurement using a plurality of lights having different wavelengths as excitation light.
[0008]
[Means for Solving the Problems]
A first aspect of the present invention comprises first and second laser light source, a laser light source selection means for selecting one of said first and second laser light sources, selected by the laser light source selection means a scanning means for scanning on the sample with a laser beam from the one laser light source as a spot light, a first photoelectric conversion means for converting the fluorescent light from the specimen into an analog electrical signal, a digital electrical said analog electrical signal A / D conversion means for converting into signals and recording means for recording the digital electric signal, and the first laser light source is selected during scanning of each pixel for each pixel scan by the scanning means switch and a second state in which the first state and the second laser light source is selected that is, first the digital electrical signal fluorescence was converted occurring in the first state, the pixel scanning every, said In recording means, wherein recorded in a first corresponding address in the first address group which has been predetermined for the laser light source, the digital second fluorescence generated in the second state is converted The electrical signal corresponds to a corresponding address in a second address group different from the first address group predetermined for the second laser light source in the recording means for each one pixel scan. is the confocal laser scanning microscope apparatus recorded.
[0009]
According to the above configuration, by changing the laser light source to be selected, the light applied to the sample is switched, and the digital electric signal corresponding to the light from the sample is recorded in advance for each laser light source in the recording means. It is recorded at the corresponding address among the predetermined addresses. This makes it possible to correctly record a digital electrical signal corresponding to the light from the sample while switching the light irradiated to the sample at a high speed using a plurality of laser light sources, and to easily obtain image information corresponding to the light from the sample. Can be built. Therefore, the confocal laser scanning microscope apparatus can be applied to measurement using a plurality of lights having different wavelengths as excitation light.
[0010]
According to a second aspect of the present invention, in the first aspect, the intensity of the laser light source is changed in synchronization with the selection of the laser light source by the laser light source selection unit.
According to this configuration, the sample is continuously irradiated with light from one laser light source by changing the intensity of the laser light source to an appropriate intensity (for example, stronger intensity) in synchronization with the selection of the laser light source. Compared to the case, it is possible to prevent the light irradiation amount of the sample per laser light source from decreasing.
[0011]
According to a third aspect of the present invention, in the first aspect, the recording means records the digital electric signal and the intensity of a laser light source that emits light that is a basis of the digital electric signal in association with each other. The configuration.
According to this configuration, based on the digital electric signal corresponding to the light from the sample and the intensity of the laser light source associated therewith, the digital electric signal obtained by correcting the luminance variation of the light emitted from the laser light source is acquired. Can do.
[0012]
According to the fourth aspect of the present invention, the first state in which the first laser light source is selected during the scanning of each pixel and the second state in which the second laser light source is selected for each pixel scan by the scanning unit. switching between states, the laser beam from said selected laser light source as a spot light scans on specimen, the fluorescence from the sample is converted into an analog electrical signal, the analog electrical signal to digital electrical signal The digital electric signal converted and converted from the first fluorescence generated in the first state is predetermined for the first laser light source on the memory for each pixel scan . The digital electric signal recorded at a corresponding address in one address group and converted from the second fluorescence generated in the second state is stored in the second memory on the memory for each one pixel scan. Predetermined for the laser light source Recorded in different second corresponding addresses in the address group from the first address group, which is a sample information recording method of the confocal laser scanning microscope apparatus.
[0013]
According to said method, the effect | action and effect similar to said 1st aspect are acquired.
The fifth aspect of the present invention is a confocal laser scanning microscope apparatus in which the first fluorescence and the second fluorescence are both incident on the same first photoelectric conversion means.
The sixth aspect of the present invention further includes a second photoelectric conversion means for obtaining the brightness of the laser beam, and the recording means includes the fluorescence obtained from the output of the first photoelectric conversion means. This is a confocal laser scanning microscope apparatus in which the digital electric signal converted from is recorded in association with the luminance signal converted from the laser light obtained from the output of the second photoelectric conversion means.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is a diagram showing a configuration example of a confocal laser scanning microscope apparatus according to an embodiment of the present invention.
[0015]
In FIG. 1, the confocal laser scanning microscope apparatus includes a microscope main body 1, a computer 2, an operation panel 3, a laser light source device 4, an image monitor 5, and the like. The device 4 and the image monitor 5 are each connected to the computer 2.
[0016]
The laser light source device 4 includes a plurality of laser light sources that can change the laser intensity, such as a semiconductor laser, a laser light source switching device 401 including an optical element such as an AOTF (acousto-optic element) or AOM / EOM, a shutter, and the like. Under the control of the computer 2, the laser light to be emitted is switched and the intensity of the laser light source that emits the laser light is changed. The laser beam used for observation or the like is set by an observer giving an instruction via the operation panel 3. Further, the switching of the laser light described above is performed by the laser light source device 4 selecting a predetermined laser light source from laser light sources that emit laser light set by an observer. That is, the laser light source device 4 switches the laser light by changing the selection target of the laser light source.
[0017]
The microscope body 1 includes an excitation dichroic mirror 101, a scanning unit 102, a revolver 103, an objective lens 104, a stage 105, a dichroic mirror 107, a lens 108, a confocal aperture 109, a photoelectric converter 110, and the like. . In the microscope main body 1, the laser light emitted from the laser light source device 4 is reflected by the excitation dichroic mirror 101 and enters the scanning unit 102. The scanning unit 102 includes a galvanometer mirror for scanning in the X-axis direction and a galvanometer mirror for scanning in the Y-axis direction, and the laser light reflected by the excitation dichroic mirror 101 is output according to a scanning control signal output from the computer 2. In addition to performing XY scanning, a scanning control end signal is output to the computer 2 for each scanning of one line by scanning in the X-axis direction. The laser light that has been XY-scanned by the scanning unit 102 is irradiated as spot light onto the observation sample 106 placed on the stage 105 via the objective lens 104 attached to the revolver 103. Light (reflected light, transmitted light, fluorescence, or the like) from the observation sample 106 by the spot light is returned to the incident optical path, reflected by the dichroic mirror 107, and collected by the lens 108. A confocal aperture 109 is disposed at a condensing position by the lens 108 to form a confocal optical system. Light that has passed through the confocal aperture 109 is input to a photoelectric converter 110 such as a photomultiplier, converted into an analog electrical signal, and input to the computer 2.
[0018]
The computer 2 includes a calculation / processing unit 2a, an A / D conversion unit 2b, a memory 2c, a recording medium (not shown) on which a control program 2d is recorded, and the like.
The A / D converter 2b converts the analog electrical signal output from the photoelectric converter 110 and corresponding to the observation sample 106 into a digital electrical signal.
[0019]
The memory 2c has a memory area for position information and an image memory area. In the memory area for position information, the scan final line position and the like are recorded. In the image memory area, an A / D converter 2b performs A / D conversion. The D-converted digital electric signal, that is, image information corresponding to the observation sample 106 is recorded. For example, image information corresponding to 1024 pixels × 768 pixels × 12 bits (4096 gradations) is recorded in the image memory area.
[0020]
The calculation / processing unit 2a is a so-called CPU (central processing unit), and reads and executes a control program 2d recorded on a recording medium (not shown) to perform the operation of the entire confocal laser scanning microscope apparatus. Control. For example, a scanning control signal is output to the scanning unit 102 in response to a scanning start instruction from the observer notified via the operation panel 3, and an analog electrical signal corresponding to the observation sample 106 output from the photoelectric converter 110. Is converted into a digital electric signal (image information) by the A / D converter 2b and transferred to the memory 2c, and the image information transferred to the memory 2c is output to the image monitor 5, and an image corresponding to the image information is output. Processing such as displaying (observation sample image) on the image monitor 5 is performed. In addition, in accordance with an instruction from the observer notified through the operation panel 3, a process for displaying a scanning menu or the like on the image monitor 5, a process for setting the gain and offset of the A / D converter 2b, Also, setting processing of the laser light source device 4 such as setting processing of the applied voltage, gain, offset, etc. of the photoelectric converter 110, various setting processing of the scanning unit 102, setting of laser light used for observation, etc. is performed.
[0021]
The operation panel 3 includes a keyboard, a pointing device such as a trackball, a joystick, or a mouse. The operation panel 3 receives various instructions from an observer and notifies the computer 2 of them. For example, a laser beam scanning start instruction, an image input instruction, a sensitivity adjustment instruction for the photoelectric converter 110, a setting instruction for a laser beam used for observation, and the like are received from the observer and notified to the computer.
[0022]
Next, the operation of the confocal laser scanning microscope apparatus configured as described above will be described.
In this operation, the confocal laser scanning microscope apparatus includes a laser light source that emits laser light having an appropriate wavelength (excitation wavelength) for each fluorescent dye on a sample stained with a plurality of fluorescent dyes. Efficient emission of the dye is performed.
[0023]
For example, for a sample stained with two types of fluorescent dyes A and B, it is appropriate for a laser light source A and a fluorescent dye B that emit laser light A having an appropriate excitation wavelength A for the fluorescent dye A. Laser beams A and B are irradiated by a laser light source B that emits a laser beam B having an excitation wavelength B, and fluorescence A ′ from the sample by the laser beam A and fluorescence B ′ from the sample by the laser beam B are generated. The fluorescent light A ′ and B ′ are incident on the photoelectric converter 110 and converted into an electric signal by being transmitted through the excitation dichroic mirror 101 and reflected by the dichroic mirror 107.
[0024]
However, when the laser light sources A and B are turned on (selected) at the same time and the sample is irradiated with the laser beams A and B at the same time, the following problems occur.
FIG. 2 is a diagram showing the wavelength characteristics of the excitation wavelengths A and B and the fluorescence wavelengths A ′ and B ′ of the fluorescence A ′ and B ′ and the dichroic mirror 101 for excitation.
[0025]
As shown in the figure, when the laser beams A and B are simultaneously irradiated, both the fluorescence A ′ having the fluorescence wavelength A ′ and the fluorescence B ′ having the fluorescence wavelength B ′ are excited by the wavelength characteristics of the dichroic mirror 101 for excitation. Fluorescent light A ′ and B ′ are incident on the photoelectric converter 110 through the dichroic mirror 101, and the luminance signal (image information) of the fluorescent light A ′ and the luminance signal (image information) of the fluorescent light B ′ are independent. Thus, there is a problem that recording (acquisition) cannot be performed.
[0026]
Therefore, in this operation, the laser light source B is controlled so that the laser light source B is turned off while the luminance signal of the fluorescent light A ′ is recorded, and the laser light source A is turned off while the luminance signal of the fluorescent light B ′ is recorded, The luminance signals of the fluorescence A ′ and B ′ can be recorded independently. That is, the laser light source selection control is performed so that the laser light source B is not selected when the laser light source A is selected, and the laser light source A is not selected when the laser light source B is selected.
[0027]
FIG. 3 is a timing chart relating to the control of the laser light source.
As shown in the figure, the laser light source A is turned ON / OFF (selected / not selected) and the laser light source B is turned OFF / ON (not selected / selected) for each pixel scan, and the fluorescence A ′ generated thereby. , B ′ are photoelectrically converted and A / D converted according to an A / D conversion clock to obtain a luminance signal corresponding to the fluorescence A ′, B ′. In the figure, D1 and D2 are luminance signals corresponding to the fluorescence A ′ and B ′ in the first pixel, and D3 and D4 are luminance signals corresponding to the fluorescence A ′ and B ′ in the second pixel. is there.
[0028]
In this way, by repeating ON / OFF of the laser light source A and OFF / ON of the laser light source B for each pixel scan until one screen scan (one frame scan) is completed, it responds to the fluorescence A ′ and B ′. The recorded image information is recorded in the memory 2c.
However, continuous luminance signals such as D1, D2, D3, D4,..., Which are image information, are recorded at different addresses on the memory 2c determined in advance for each laser light source. For example, the luminance signal from the laser light source A such as D1, D3,... Is recorded in the address of the odd address previously determined for the laser light source A, and the luminance signal from the laser light source B such as D2, D4,. The address is recorded at an even address determined for the laser light source B. Alternatively, the luminance signal from the laser light source A such as D1, D3,... Is recorded at consecutive addresses starting from aaa predetermined for the laser light source A, and the luminance signal from the laser light source B such as D2, D4,. , Recording is performed at consecutive addresses starting from bbbb predetermined for the laser light source B. As a result, image information for one screen corresponding to each fluorescence can be easily constructed based on the luminance signal recorded at each address.
[0029]
As described above, according to the present embodiment, the luminance signal corresponding to the light from the sample is recorded at the corresponding address on the memory that is determined in advance for each laser light source. The luminance signal corresponding to the light from the sample can be correctly recorded while switching to, and image information for one screen corresponding to each light from the sample can be easily constructed. Therefore, the confocal laser scanning microscope apparatus can be applied to measurement using a plurality of different lights as excitation light.
[0030]
In this embodiment, the case where two types of fluorescence are observed has been described as an example. However, even when three or more types of fluorescence are observed, the luminance signal corresponding to each fluorescence of each pixel is previously observed. Image information corresponding to each fluorescence can be correctly acquired by recording at a corresponding address on a memory determined for each laser light source that emits a laser beam to be used.
[0031]
Further, in the present embodiment, as shown in the timing chart of FIG. 3, when two types of laser light sources are turned on / off within one pixel scanning to switch between the two types of laser light within one pixel scanning, The amount of laser light applied to the sample is halved compared to when switching is not performed. Therefore, in each A / D conversion performed in synchronization with the A / D conversion clock, the intensity of the laser light source is changed to an appropriate intensity so as to optimize the light amount of the laser light irradiated on the sample. Also good. For example, when the above-mentioned two types of laser light are used by switching, the intensity of the laser light source is changed to twice the intensity when using one type of laser light without switching. good. By doing so, the decrease in the amount of light can be corrected. Since the A / D conversion clock is synchronized with ON / OFF (selected / not selected) of the laser light source, the intensity change of the laser light source is also performed in synchronization with the ON / OFF of the laser light source. . Further, it goes without saying that such intensity change of the laser light source is performed in the same manner when three or more types of laser light are switched and used.
[0032]
In the present embodiment, a photoelectric conversion unit for acquiring the luminance of the laser light emitted from the laser light source device 4 is newly provided, and when a luminance signal corresponding to the light from the sample is recorded in the memory 2c, A luminance signal corresponding to the laser beam that is the output of the photoelectric conversion unit may also be recorded in association with it. That is, the luminance signal corresponding to the light from the sample and the intensity of the laser light source that emitted the laser light that is the basis thereof may be recorded in association with each other. By doing in this way, the luminance signal according to the light from the sample obtained by the luminance fluctuation of the laser light emitted from the laser light source device 4 can be corrected, and the laser emitted from the laser light source device 4 Information from the sample can be obtained more accurately than removing light noise. For example, if it is determined that the output of the laser beam has decreased by 10% from the luminance signal corresponding to the recorded laser beam in association with the luminance signal Da of an arbitrary pixel, the luminance signal of the arbitrary pixel By newly recording a value obtained by multiplying Da by 1.1, the luminance signal can be corrected. Moreover, you may make it display it on the image monitor 5 as needed.
[0033]
The confocal laser scanning microscope apparatus and the sample information recording method of the present invention have been described in detail above. However, the present invention is not limited to the above-described embodiment, and various improvements and modifications can be made without departing from the gist of the present invention. Of course, you may go.
[0034]
【The invention's effect】
As described above in detail, according to the present invention, it is possible to correctly record luminance information corresponding to light from the sample while switching a plurality of laser beams at high speed, and to respond to each light from the sample. Therefore, the confocal laser scanning microscope apparatus can be applied to measurement using a plurality of different light beams as excitation light.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration example of a confocal laser scanning microscope apparatus according to an embodiment of the present invention.
FIG. 2 is a diagram showing the fluorescence wavelengths A ′ and B ′ of the excitation wavelengths A and B and the fluorescence A ′ and B ′ and the wavelength characteristics of the excitation dichroic mirror.
FIG. 3 is a timing chart relating to control of a laser light source.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Microscope main body 2 Computer 2a Calculation / processing part 2b A / D conversion part 2c Memory 2d Program 3 Operation panel 4 Laser light source apparatus 5 Image monitor 101 Excitation dichroic mirror 102 Scan unit 103 Revolver 104 Objective lens 105 Stage 106 Observation sample 107 Dichroic Mirror 108 Lens 109 Confocal aperture 110 Photoelectric converter

Claims (6)

First and second laser light sources;
A laser light source selection means for selecting one of said first and second laser light sources,
A scanning means for scanning on the sample with a laser beam from the one laser light source selected by the laser light source selection means as a spot light,
A first photoelectric conversion means for converting the fluorescent light from the specimen into an analog electrical signal,
A / D conversion means for converting the analog electric signal into a digital electric signal;
Recording means for recording the digital electrical signal;
With
For each one pixel scan by the scanning means, switching between a first state in which the first laser light source is selected and a second state in which the second laser light source is selected during scanning of each pixel,
The digital electric signal obtained by converting the first fluorescence generated in the first state is a predetermined first value for the first laser light source in the recording means for each one pixel scan . Recorded at the corresponding address in the address group ,
The digital electric signal obtained by converting the second fluorescence generated in the second state is predetermined for the second laser light source in the recording unit for each pixel scan. Recorded in a corresponding address in a second address group different from the address group of
A confocal laser scanning microscope apparatus. Changing the intensity of the laser light source in synchronization with the selection of the laser light source by the laser light source selection means,
The confocal laser scanning microscope apparatus according to claim 1. The recording means records the digital electric signal and the intensity of a laser light source that emits light that is the basis of the digital electric signal in association with each other.
The confocal laser scanning microscope apparatus according to claim 1. For each one pixel scan by the scanning means, the first state in which the first laser light source is selected during the scanning of each pixel and the second state in which the second laser light source is selected are switched ,
The laser beam from said selected laser light source as a spot light scans on specimen,
The fluorescence from the sample is converted into an analog electrical signal,
Converting the analog electrical signal into a digital electrical signal;
The digital electric signal obtained by converting the first fluorescence generated in the first state is a predetermined first address for the first laser light source on the memory for each one pixel scan. Record at the corresponding address in the group ,
The digital electric signal obtained by converting the second fluorescence generated in the second state is predetermined for the second laser light source on the memory for each pixel scan. Recording at a corresponding address in a second address group different from the address group of
A sample information recording method for a confocal laser scanning microscope apparatus. The first fluorescence and the second fluorescence are both incident on the same first photoelectric conversion means,
The confocal laser scanning microscope apparatus according to claim 1. Furthermore, it includes a second photoelectric conversion means for obtaining the luminance of the laser beam,
In the recording means, the digital electric signal obtained by converting the fluorescence obtained from the output of the first photoelectric conversion means and the luminance obtained by converting the laser light obtained from the output of the second photoelectric conversion means Recorded in association with the signal,
The confocal laser scanning microscope apparatus according to claim 1.

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