JP3855419B2 - Laser microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a laser microscope that enables observation of a sample image by detecting light generated by irradiating a sample with laser light as excitation light.
[0002]
[Prior art]
FIG. 4 is a diagram for explaining an outline of a laser scan type confocal microscope system which is an example of a conventional laser microscope.
[0003]
In this confocal microscope system, when performing fluorescence observation by irradiating a sample with a single excitation light, among the excitation light generated from the laser light source 1 by the excitation filter unit 2, the optimum wavelength for the fluorescent dye attached to the sample And the excitation light condensed on the sample surface 4 by the objective lens 3 is scanned two-dimensionally using the scanner 5. Then, the fluorescence generated from the fluorescent dye attached to the sample surface 4 and reversing the optical path of the excitation light is separated by, for example, the dichroic mirror 11 and then detected by the fluorescence detector 6, and its analog output is detected by the A / D converter 7. A digital signal is used, and an image of the sample is created using the computer 8.
[0004]
At this time, if necessary, transmitted light that has passed through the sample out of the excitation light irradiated on the sample surface 4 is detected by the transmitted light detector 9, and its analog output is also digitally converted by the A / D converter 10 in the same manner as fluorescence detection. And a transmission image is created using the computer 8.
[0005]
When observing a sample by multiple excitation with a plurality of fluorescence wavelengths, fluorescent dyes having different absorption wavelengths and fluorescence wavelengths are used. The excitation light of a plurality of wavelengths corresponding to such a fluorescent dye is selected by the excitation filter unit 2 and supplied to the sample surface 4, and a plurality of fluorescence corresponding to each wavelength returned from the fluorescent dye on the sample surface 4 is obtained. , Separated by the dichroic mirrors 11, 12, and 13, detected by the fluorescence detectors 6, 14, and 15 provided for each wavelength, and analog signals from the respective fluorescence detectors 6, 14, and 15 are converted into A / D converters 7, 16 and 17 are digitized and the computer 8 forms an image. In the transmission image observation, the transmitted light that has passed through the sample surface 4 illuminated with a plurality of excitation lights is detected by the transmitted light detector 9, and an image is created by the computer 8 as described above.
[0006]
[Problems to be solved by the invention]
In transmission image observation when performing multiple excitation as described above, all transmitted excitation light is detected, but the stability of the laser output varies depending on the wavelength of the laser light used for excitation. For this reason, when laser light having a wavelength with poor stability is included, the detection output from the transmitted light detector 9 is not stable, and the fluctuation of the laser output appears as fluctuation (noise) of the intensity of the pixel in the observation image. .
[0007]
The present invention has been made in view of such conventional problems, and an object of the present invention is to provide a laser microscope that can reduce fluctuations in pixel intensity in an observation image in transmission image observation at the time of multiple excitation. And
[0008]
[Means for Solving the Problems]
In order to solve the above problems, a laser microscope according to the present invention includes a light source that generates a plurality of laser beams having different wavelengths, and an illumination optical system that supplies the plurality of laser beams from the light source to a sample via an objective lens. And an observation system for obtaining a sample image by acquiring fluorescence or reflected light emitted from the sample by irradiation of the plurality of laser beams, and detecting a laser beam transmitted through the sample illuminated by the illumination optical system, A transmitted light detection device for obtaining a sample image, a wavelength selection device for selecting a wavelength of laser light incident on the transmitted light detection device, a storage device for storing intensity fluctuation amounts for each wavelength component of the plurality of laser beams, and A determination device for determining a wavelength corresponding to the smallest intensity fluctuation amount among the intensity fluctuation amounts for each wavelength component; and a control device for controlling the wavelength selection device so as to select the determined wavelength; Characterized in that it comprises a.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, specific embodiments of a laser microscope according to the present invention will be described with reference to the drawings.
[0012]
[First Embodiment]
FIG. 1 is a block diagram showing a basic structure of a confocal microscope system that is a laser microscope of the first embodiment.
[0013]
In this confocal microscope system, as an illumination optical system, a laser light source 1 that generates a plurality of excitation lights necessary for observation, and an excitation for selecting an optimum wavelength for a fluorescent dye used for observation from these excitation lights. The filter unit 2, the separation dichroic mirror 21 that reflects the excitation light that has passed through the excitation filter unit 2, the scanner 5 for scanning the excitation light on the sample surface 4, and the sample surface by condensing the excitation light 4 and an objective lens 3 for projecting onto 4.
[0014]
Here, the laser light source 1 is, for example, a KrAr laser capable of outputting three wavelengths. This KrAr laser can output excitation light having wavelengths of 488 nm, 568 nm, and 647 nm. These excitation lights enter the excitation filter unit 2 through the mirror 20. The excitation filter unit 2 can replace a plurality of band-pass filters on the optical path for selectively transmitting necessary excitation light of one wavelength or more of the excitation light according to the type of fluorescent dye attached to the sample. It can be arranged. As the excitation filter, one wavelength, two wavelengths, and three wavelengths (arbitrary combinations) can be selected. When using three wavelengths, a filter that transmits three wavelengths is selected.
[0015]
In addition to the objective lens 3 serving as a component of the above-described illumination optical system, the scanner 5 and the separating dichroic mirror 21 as an observation system, the confocal microscope system collects light by the objective lens 3 and is scanned by the scanner 5. The lens 22 for once condensing the fluorescence that has been descanned by the first dichroic mirror 21, the pinhole 23, and the first to third dichroic that divides the excitation light that has passed through the pinhole 23 for each fluorescence wavelength First to third fluorescence detectors 6, 14, and 15 that individually detect and convert fluorescence for each wavelength divided by the mirrors 11, 12, and 13 and the first to third dichroic mirrors 11, 12, and 13. With.
[0016]
In addition, this confocal microscope system, as a transmitted light detection system, photoelectrically converts transmitted light and fluorescence by an imaging lens 91 that collects transmitted light that has passed through the sample surface 4 and fluorescence generated from the sample surface 4. And a transmitted light detector 9 for detection.
[0017]
In addition, this confocal microscope system is disposed between the sample surface 4 and the transmitted light detector 2 as the wavelength selection unit 40, and only emits light of, for example, 488 nm out of excitation light and fluorescence composed of three wavelength components. A band-pass filter 41 for transmission is provided. The band-pass filter 41 is movable between an operating position (solid line) on the optical path of transmitted light and a retracted position (dotted line) retracted from the optical path of transmitted light as shown in the figure. When the bandpass filter 41 is moved to the operating position, it is possible to prevent excitation light of other wavelengths (568 nm, 647 nm) of the KrAr laser that is the laser light source 1 from entering the transmitted light detector 9.
[0018]
Furthermore, this confocal microscope system is an A / D converter 7 that digitizes electrical detection signals from the first to third fluorescence detectors 6, 14, 15 and the transmitted light detector 9 as a processing control device, 16, 17, and 10, and a computer 80 that performs an appropriate process on the digital signals from these A / D converters 7, 16, 17, and 10 to image and display the sample state. The computer 80 controls the excitation filter unit 2 to appropriately set the wavelength of excitation light that is incident on the sample surface 4. The computer 80 controls the operation of the scanner 5 so that the excitation light is scanned on the sample surface 4 at a necessary interval and speed. The control signal for the scanner 5 is referred to when the digital signals from the A / D converters 7, 16, 17, and 10 are imaged. In addition, the computer 80 controls the wavelength selection unit 40 to move the bandpass filter 41 to either the operating position indicated by the solid line or the retracted position indicated by the dotted line. When the bandpass filter 41 is moved to the operating position indicated by the solid line, only stable excitation light having a wavelength of 488 nm of the KrAr laser that is the laser light source 1 is incident on the transmitted light detector 9, and noise of light having wavelengths of 568 nm and 647 nm. Can be prevented from entering the detector 9 for transmitted light.
[0019]
The operation of the apparatus of FIG. 1 will be briefly described. In this confocal microscope system, a fluorescent dye having an absorption wavelength corresponding to the wavelength (488 nm, 568 nm, 647 nm) of the KrAr laser light from the laser light source 1 is attached to the sample when performing fluorescence observation. Then, the excitation light from the laser light source 1 is incident on the sample disposed on the sample surface 4 via the scanner 5 and the objective lens 3 in a spot shape, and the spot-like excitation light is scanned on the sample surface 4. The first to third detectors 6, 14, and 15 detect the fluorescent light generated from the fluorescent dye existing on the sample surface 4 and reverse, and the analog output is digitally converted by the A / D converters 7, 16, and 17. And an image of the sample is created using the computer 80.
[0020]
At this time, transmitted light that passes through the sample surface 4 in the excitation light is detected by the transmitted light detector 9, and its analog output is digitized by the A / D converter 10 in the same manner as fluorescence detection, and transmitted using the computer 80. An image is also created. The KrAr laser light from the laser light source 1 often has different noise levels at each wavelength, and the light at 488 nm is often the most stable. Therefore, when observing transmitted light at a plurality of wavelengths including 488 nm, the band-pass filter 41 is disposed at the operating position on the optical path of the transmitted light, and only 488 nm light of the plurality of excitation lights reaches the transmitted light detector 9. To do. By doing so, noise of light of other wavelengths (568 nm, 647 nm) of the KrAr laser can be removed, and transmission image observation with less noise can be performed.
[0021]
[Second Embodiment]
Hereinafter, the confocal microscope system according to the second embodiment will be described with reference to FIG. Note that the apparatus of the second embodiment is a modification of the apparatus of the first embodiment, and the same portions are denoted by the same reference numerals and redundant description is omitted.
[0022]
The confocal microscope system according to the second embodiment is a modification of the apparatus according to the second embodiment. The computer 80 is used to calculate the noise level from the digital image obtained from the output of the transmitted light detector 9, This is a system that automatically determines the most suitable bandpass filter for transmission image observation.
[0023]
The wavelength selection filter unit 140 includes a plurality of band pass filters 141, 142, and 143 that selectively transmit a plurality of excitation lights from the laser light source 1. If any of these bandpass filters 141, 142, 143 is arranged on the optical path, only the excitation light corresponding to the transmission wavelength can be supplied to the transmitted light detector 9. If all the bandpass filters 141, 142, 143 are retracted from the optical path, all the excitation light can be supplied to the transmitted light detector 9.
[0024]
Hereinafter, the operation of the confocal microscope system of the second embodiment will be described with reference to the flowchart of FIG. In advance, only a cover glass is placed on the sample surface 4 as a sample, and the detection output from the transmitted light detector 9 is set to correspond to the laser output. Then, the wavelength selection filter unit 140 is operated from the computer 80 side, and any one of the bandpass filters 141, 142, 143 is arranged on the optical path (step S1). As a result, only single-wavelength excitation light can be selected and incident on the transmitted light detector 9. Next, the transmitted light is detected while scanning the selected excitation light on the sample surface 4, and noise is measured (steps S2 and S3). As a noise measurement method at this time, for example, the standard deviation of the luminance distribution in the entire transmission digital image is calculated. If the obtained standard deviation is small, it can be considered that the intensity fluctuation amount, that is, noise is small. Next, it is determined whether or not noise measurement has been completed for all excitation wavelengths (step S4). If noise measurement has not been completed for all excitation wavelengths, the process returns to step S1 to place another bandpass filter on the optical path, and noise measurement is performed again in steps S2 and S3. When noise measurement has been completed for all excitation wavelengths, the noise levels at each excitation wavelength are ranked based on the obtained standard deviation, and such ranking is stored in a storage device provided in the computer 80. (Step S5). When observing the sample, the sample is placed on the sample surface 4 and the user inputs an excitation wavelength corresponding to the currently used fluorescent dye to the computer 80. The computer 80 controls the excitation filter unit 2 to select a corresponding bandpass filter (step S6). Next, the computer 80 determines the excitation wavelength with the least noise among the wavelengths used for observation, controls the wavelength selection filter unit 140 to select a bandpass filter that transmits this excitation wavelength, and this excitation wavelength. A transmission image corresponding to is automatically created (step S7). Thereby, it is possible to observe a transmission image with little fluctuation of pixel intensity, that is, with less noise.
[0025]
Note that the noise level ranking at each excitation wavelength is stored once in the memory of the computer 80, and when observing the transmitted image thereafter, the noise level ranking processing (until the laser light source 1 is replaced) ( It is not necessary to repeat steps S1 to S4). However, by regularly performing the noise level ranking at each excitation wavelength, even if the noise level varies due to the characteristic variation of the laser light source 1, this can be dealt with.
[0026]
As described above, the present invention has been described according to the embodiment, but the present invention is not limited to the above embodiment. For example, in the above transmission image observation, normal absorption is observed. However, when observation is performed with a general optical microscope such as differential interference or phase difference, noise from a plurality of transmission excitation lights is also observed. Only those with a small amount can be selected and detected.
[0027]
Further, not only a single transmission wavelength but also a plurality of relatively stable wavelengths can be selected to detect a transmission image.
[0028]
【The invention's effect】
In order to solve the above-mentioned problems, according to the laser microscope of the present invention, a transmitted light detection device that detects the laser light transmitted through the sample illuminated by the illumination optical system and obtains the sample image, and the transmitted light detection And a wavelength selection device that selects the wavelength of the laser beam incident on the device, so that even if the laser beam with poor stability is included in the plurality of laser beams supplied to the sample, A transmission image with less noise can be obtained by cutting the wavelength component with the wavelength selection device.
[0029]
According to a preferred aspect, the laser further supplied to the sample is provided with a control device that controls wavelength selection by the wavelength selection device based on information on the wavelength of the laser light supplied from the light source to the sample. It is possible to select an appropriate wavelength according to the change of the light and guide only the laser beam corresponding to this wavelength to the transmitted light detection device.
[0030]
According to a preferred embodiment, the control device stores a storage device that stores intensity fluctuation amounts for each wavelength component of the plurality of laser beams generated by the light source, and a laser beam supplied from the light source to the sample. A determination device for determining a wavelength corresponding to the smallest intensity fluctuation amount among the corresponding intensity fluctuation amounts, so that an optimum wavelength can be easily selected according to a change in the laser beam supplied to the sample, etc. Only the laser beam corresponding to the wavelength can be guided to the transmitted light detection device.
[Brief description of the drawings]
FIG. 1 is a block diagram illustrating the structure of a confocal microscope system according to a first embodiment.
FIG. 2 is a block diagram illustrating the structure of a confocal microscope system according to a second embodiment.
FIG. 3 is a flowchart for explaining the operation of the confocal microscope system of FIG. 2;
FIG. 4 is a diagram illustrating the structure of a conventional confocal microscope system.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Laser light source 2 Excitation filter unit 3 Objective lens 4 Sample surface 5 Scanner 7, 16, 17, 10 A / D converter 6,14,15 Fluorescence detector 9 Transmitted light detector 11,12,13 Dichroic mirror 6,14, 15 Fluorescence detector 21 Separation dichroic mirror 22 Lens 23 Slit 40, 140 Wavelength selection unit 41, 141, 142, 143 Band pass filter 80 Computer 91 Imaging lens

Claims (1)

A light source that generates a plurality of laser beams having different wavelengths from each other;
An illumination optical system that supplies the plurality of laser beams from the light source to a sample via an objective lens;
An observation system for obtaining a sample image by acquiring fluorescence or reflected light emitted from the sample by irradiation with the plurality of laser beams;
A transmitted light detection device for obtaining a sample image by detecting laser light transmitted through the sample illuminated by the illumination optical system;
A wavelength selection device for selecting a wavelength of laser light incident on the transmitted light detection device;
A storage device for storing an intensity fluctuation amount for each wavelength component of the plurality of laser beams;
A determination device for determining a wavelength corresponding to the smallest intensity fluctuation amount among the intensity fluctuation amounts for each wavelength component;
A control device for controlling the wavelength selection device to select the determined wavelength;
A laser microscope comprising:

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