JP4331454B2 - Scanning laser microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a laser scanning microscope.
[0002]
[Prior art]
In recent years, it is desired to measure various detection factors as the lineup of fluorescent reagents increases and the labeling state changes. In addition to the fluorescence intensity, they efficiently detect the fluorescence spectrum and deflection characteristics, and quantification as acquired data is also desired. Furthermore, miniaturization as an apparatus is also expected.
[0003]
A scanning laser microscope using an acousto-optic element (Acousto-Optic Tunable Filter: hereinafter referred to as “AOTF” in the present specification) is known (see Japanese Patent Laid-Open No. 2001-124997).
[0004]
In this scanning laser microscope, an optical member is connected behind the spectrum selective element (AOTF). And excitation light can be selected by injecting a laser beam from the 1st-order diffracted light path of AOTF. As a result, the AOTF functions as a highly efficient beam splitter for guiding the fluorescence from the microscope to the detector on the optical path of the 0th-order diffracted light. Furthermore, this JP-A-2001-124997 has the following description. Using the dispersibility or birefringence of AOTF, fluorescence can be separated into, for example, two different polarization components. By detecting each of the two deflection components, a scanning polarization laser microscope that does not require a polarizer or an analyzer can be obtained.
[0005]
However, the combination of the AOTF and the optical member according to Japanese Patent Laid-Open No. 2001-124997 can select the wavelength in accordance with the excitation wavelength of the laser, but does not realize the spectral effect that makes the wavelength selection for fluorescence. Further, although the polarization component separation is achieved by statically using the birefringence of the prism of AOTF, the acoustooptic effect of AOTF is not actively used. For this reason, in Japanese Patent Laid-Open No. 2001-124997, it is not possible to detect a polarization component of fluorescence while selecting a wavelength (that is, while performing spectroscopy).
[0006]
Therefore, in order to select a wavelength in Japanese Patent Laid-Open No. 2001-124997, it is necessary to place a multiband detector, a spectrometer such as a grating spectrometer, and a prism spectrometer further behind the AOTF and the optical member. Increasing the size is inevitable.
[0007]
A microscope using AOTF is known (see USP 5,841,577).
[0008]
In this microscope, the AOTF is disposed in the illumination optical path. Then, by superimposing two outgoing lights having different polarization components selected by the AOTF and using the combining means again as one excitation light, the excitation light can be efficiently illuminated on the sample. Further, by arranging an AOTF on the observation side, it is possible to observe fluorescence from a sample wavelength-selected by the AOTF with a CCD camera. Further, US Pat. No. 5,841,577 also discloses a method of using two AOTFs to superimpose separated light components having different polarization components again as a light beam and a method of performing epi-illumination fluorescent illumination using a dichroic mirror. Are listed.
[0009]
However, US Pat. No. 5,841,577 relates to an illumination method for fluorescence observation, and is not configured to acquire spectrum data while selecting a wavelength with a scanning laser microscope. By providing an AOTF in the observation optical path, fluorescence observation can be performed with a CCD camera. However, when applied to a scanning laser microscope, only the polarization component contained in one primary diffracted light is detected. There is a problem that light loss and the fluorescence intensity of the actual sample due to this cannot be correctly reflected.
[0010]
Further, in US Pat. No. 5,841,577, in order to block undesired zero-order diffracted light, a condenser lens for dark field, a member serving as a stopper on the optical axis, or two separated light beams are again combined into one. It is necessary to use a combining means or the like that superimposes the excitation light, which inevitably increases the size of the apparatus.
[0011]
[Problems to be solved by the invention]
It is an object of the present invention to provide a scanning laser microscope that can be simplified in configuration, facilitated in miniaturization, can perform high-efficiency spectroscopy, and can also realize high-speed and high-resolution spectral detection. To do.
[0012]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present invention has taken the following measures.
[0013]
The scanning laser microscope according to the first aspect of the present invention includes an objective lens that focuses laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning and an acoustooptic that is arranged on the optical path of the fluorescent or reflected light and selectively deflects only light having a wavelength corresponding to the frequency of the high-frequency voltage applied from the incident fluorescent or reflected light And an optical detection unit that detects light passing through the acoustooptic device, and a frequency scanning unit that switches and sets a frequency of a high-frequency voltage applied to the acoustooptic device.
[0014]
A scanning laser microscope according to a second aspect of the present invention includes an objective lens that collects laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning, an acoustooptic element arranged on the optical path of the fluorescence or reflected light, and the acoustooptic element, when a high frequency voltage is applied, the high frequency of the incident fluorescence or reflected light Light having a wavelength corresponding to the frequency of the voltage is diffracted into + 1st order diffracted light and −1st order diffracted light, and when the high frequency voltage is not applied, the incident fluorescence or reflected light is transmitted as 0th order diffracted light, and the acoustooptic A high-frequency signal control unit that controls on / off of application of the high-frequency voltage to the element and sets the high-frequency voltage, a first photodetector that receives the + 1st-order diffracted light, and the -1st-order diffraction Characterized by comprising a second photodetector for receiving the.
[0015]
A scanning laser microscope according to a third aspect of the present invention includes an objective lens that focuses laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning, an acoustooptic element arranged on the optical path of the fluorescence or reflected light, and the acoustooptic element, when a high frequency voltage is applied, the high frequency of the incident fluorescence or reflected light Light having a wavelength corresponding to the frequency of the voltage is diffracted into + 1st order diffracted light and −1st order diffracted light, and when the high frequency voltage is not applied, the incident fluorescence or reflected light is transmitted as 0th order diffracted light, and the acoustooptic A high-frequency signal control unit that controls on / off of application of the high-frequency voltage to the element and sets the high-frequency voltage, a first photodetector that receives the + 1st-order diffracted light, and the -1st-order diffraction A second photodetector for receiving the light, and the laser light source transmits the acoustooptic element without being modulated and guided to the objective lens so as to be guided to the objective lens. It is arranged in the optical path.
[0016]
A scanning laser microscope according to a fourth aspect of the present invention includes an objective lens that condenses laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning, an acoustooptic element arranged on the optical path of the fluorescence or reflected light, and the acoustooptic element, when a high frequency voltage is applied, the high frequency of the incident fluorescence or reflected light While diffracting light having a wavelength corresponding to the frequency of the voltage into first-order diffracted light and not applying the high-frequency voltage, the incident fluorescence or reflected light is transmitted as zero-order diffracted light, and the high-frequency voltage applied to the acoustooptic device A high-frequency signal control unit that controls the on / off of the application of light and sets the high-frequency voltage; and a photodetector that receives the first-order diffracted light. Sweeping the frequency of the high-frequency voltage, by obtaining the output signal of the photodetector at each frequency, to obtain the spectral data of the fluorescence or the reflected light.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described with reference to the drawings.
[0018]
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
[0019]
(First embodiment)
FIG. 1 is a diagram showing a main part of a microscope according to the first embodiment of the present invention. In FIG. 1, the scanning unit 20 is provided with a collimator lens 21. Laser light oscillated from the laser light source 22 is guided to the collimator lens 21 through the fiber 23. The laser light guided to the scanning unit 20 is converted into parallel light by the collimator lens 21 and guided to the reflected light path of the beam splitter 24. FIG. 1 shows the basic configuration of the system. In order to give the laser light source 22 a plurality of wavelengths, a multi-line laser or a plurality of laser light sources may be used. A plurality of dichroic mirrors 24 may be provided.
[0020]
Here, it is assumed that the wavelength of the laser light oscillated from the laser light source 22 is 488 nm of the Ar laser, and the wavelength characteristic of the beam splitter 24 reflects 488 nm and transmits wavelengths longer than 488 nm (500 nm to 700 nm). Assume that The 488 nm laser light reflected by the beam splitter 24 is deflected by optical scanning mirrors 251 and 252 which are galvanometer scanners constituting the optical scanning means 25. The laser beam deflected by the optical scanning mirror 252 is further condensed and irradiated on the sample 27 by the objective lens 26 via the pupil projection lens 261 and the imaging lens 262 of the microscope.
[0021]
The optical scanning mirrors 251 and 252 perform scanning control of the laser light so that the laser light is deflected in directions orthogonal to each other, and the light spot focused on the sample 27 scans the sample two-dimensionally. By irradiating a laser beam of 488 nm, fluorescence having a wavelength longer than 488 nm emitted from the sample travels in the opposite direction and the same optical path as the laser beam (that is, the objective lens 26, the imaging lens 262, and the pupil projection lens 261). Then, the light is reflected from the optical scanning mirrors 252 and 251 and guided to the transmission optical path of the beam splitter 24.
[0022]
The fluorescence guided to the transmission optical path of the beam splitter 24 is reflected by the reflection mirror 19 and then imaged on the confocal pinhole 29 by the confocal lens 28. The fluorescence transmitted through the confocal pinhole 29 is converted into parallel light by the lens 30. On the optical path of the parallel light, an acousto-optic element unit 31 constituting a frequency scanning unit is disposed via an optical path switching mirror 32.
[0023]
The optical path switching mirror 32 is disposed so as to be retractable from a position 32a on the optical path of parallel light to a position 32b indicated by a broken line in the drawing. The optical path switching mirror 32 is retracted to the position 32b, and the fluorescence guided to the optical path transmits the light in the fluorescence wavelength region and passes through the barrier filter 33 that cuts the laser wavelength (here, 488 nm). The light quantity is detected by being guided to a photodetector 34 having a photomultiplier tube.
[0024]
The photodetector 34 is a detector for acquiring a scanning image of a normal scanning laser microscope. The photodetector 34 sequentially detects the amount of fluorescent light corresponding to one light spot in synchronization with the scanning of the optical scanning mirrors 251 and 252. Then, a two-dimensional image is obtained by displaying the light quantity at each light spot position on each pixel of a monitor (not shown). The two-dimensional image obtained here is an image showing the total amount of light in the wavelength range extracted by the barrier filter 33 among the fluorescence emitted from each light spot position, and includes fine wavelength information such as spectral characteristic data. Absent.
[0025]
The acoustooptic device unit 31 includes, for example, an acoustooptic device 311 (hereinafter referred to as “AOTF”) and a photodetector (photomultiplier tube) 312 for measuring spectrum characteristics as a light detecting means. The AOTF 311 usually includes a crystal (not shown) and an RF transducer. When a high frequency voltage (RF) having a desired frequency is applied to the RF transducer, a sound wave having a desired frequency is generated in the crystal. Only the wavelength satisfying the Bragg reflection condition between the RF frequency and the incident light wavelength is deflected as the first-order diffracted light and detected by the photodetector 312. Note that a device other than the PMT capable of detecting a signal by photoelectric conversion may be used as the detector 312 to obtain an equivalent effect.
[0026]
The wavelength width of the light deflected as the first-order diffracted light at one RF frequency is usually 3 nm or less, and the wavelengths of the other light are guided through the AOTF 311 crystal to the optical path. Therefore, by sequentially switching or scanning the RF frequency and measuring the amount of light detected by the photodetector 312 for each frequency, the spectral characteristics of the fluorescence can be detected.
[0027]
Here, the control unit 40 shown in FIG. 1 controls the optical scanning mirrors 251 and 252, the photodetector 34, the photodetector 312, and the RF transducer based on command information (not shown). The control unit 40 has the following three main functions.
[0028]
(1) A function of detecting the amount of light incident on the photodetector 34 and the photodetector in synchronization with (in synchronization with) the scanning timing of the optical scanning mirrors 251 and 252.
[0029]
(2) A function of detecting the light amounts of the photodetector 34 and the photodetector 312 in synchronization with (in synchronization with) the scanning frequency of the RF frequency.
[0030]
(3) The RF frequency of the RF transducer is scanned (synchronized) with the scanning timing of the optical scanning mirrors 251 and 252 and the RF frequency of the RF transducer is scanned (synchronized). A function of detecting the light quantity of the photodetector 312.
[0031]
An operation of measuring spectral characteristic data with the acoustooptic device unit 31 in the above configuration will be described. First, when using the acoustooptic device unit 31, the optical path switching mirror 32 is moved to the position 32a by a switching mechanism (not shown). As a result, the laser light emitted from the laser light source 22 is deflected by the optical scanning mirrors 251 and 252. As a result, when a light spot is formed at an arbitrary point on the sample 27, the fluorescence emitted from the sample 27 passes through the confocal pinhole 29 via the beam splitter 24 and the like. The fluorescence transmitted through the confocal spot 29 is converted into parallel light by the lens 30, is reflected by the optical path switching mirror 32, and enters the AOTF 311.
[0032]
Here, it is assumed that the relationship between the RF frequency and the wavelength satisfying the Bragg reflection condition is as follows. It is assumed that the RF frequency decreases by 1 MHz every time the wavelength of 4 nm becomes longer, 132 MHz at 492 nm, 131 MHz at 496 nm, and 130 MHz at 500 nm. Note that the relationship between the RF frequency and the wavelength may actually be a non-linear relationship due to a difference in refractive index depending on the wavelength of the crystal of the AOTF 311, but here, it will be described as a linear relationship for simplification.
[0033]
When the RF frequency of 132 MHz is applied to the crystal by the RF transducer in the AOTF 311, light having a wavelength corresponding to this frequency, 492 nm, is deflected as the first-order diffracted light. As a result, the intensity of light having a wavelength near 492 nm is detected by the photodetector 312. Then, when the RF frequency is applied to the crystal at a frequency corresponding to the wavelength of 496 nm and 131 MHz is applied to the crystal, the light of 496 nm is deflected as the first-order diffracted light. As a result, the intensity of the wavelength near 496 nm is detected by the photodetector 312.
[0034]
Hereinafter, similarly, the RF frequency is sequentially switched (scanned) up to 90 MHz corresponding to the RF frequency of 660 nm every 1 MHz, and the light intensity of the wavelength corresponding to each frequency is detected by the photodetector 312. Detect with. Through the above steps, as shown in FIG. 2, spectral characteristic data of fluorescence from 492 nm to 660 nm is acquired. The spectral characteristic data obtained here is data of fluorescence emitted from one point on the sample to which the light spots are connected.
[0035]
When obtaining spectral characteristic data from a wide range (scanned image acquisition range) of the sample, the optical scanning mirrors 251 and 252 are scanned, and the light spots tied on the sample 27 are sequentially scanned. The above-described steps for obtaining fluorescence spectral characteristic data from one light spot (RF frequency scanning and light amount measurement at a wavelength corresponding to each RF frequency by the photodetector 312) are performed. At this time, the control unit 40 controls the optical scanning mirrors 251 and 252, the frequency scanning of the RF transducer, and the light measurement by the photodetector 312 in synchronization.
[0036]
Here, the acquisition time of spectral characteristic data for each pixel will be considered. For example, in an image of 512 × 512 pixels, the detection time with a photodetector of one wavelength corresponding to one RF frequency is 1 μsec, and the switching time of the RF frequency is 1 μsec. Since the RF frequency is switched from 132 MHz to 90 MHz every 1 MHz, the data acquisition time for one pixel is (132−90) × 2 + 1 = 85 μsec, and the data acquisition time for all pixels is 85 (μsec). ) × 512 × 512 = 22.3 sec. Data is acquired within 30 seconds even if allowances such as the blanking period of the optical scanning mirrors 251 and 252 are taken into account.
[0037]
In the above description, the case where the RF frequency is changed to 1 MHz and the measurement is performed from 492 nm to 660 nm in increments of 4 nm has been described. However, the RF frequency depends on the wavelength resolution of the AOTF 311 and the overall data acquisition time. Needless to say, it may be determined appropriately in consideration of the spectral range, step size, and the like. Needless to say, the reason why the data is acquired in the wavelength range longer than 488 nm of the laser wavelength, which is the excitation light, is that the fluorescence is emitted at a wavelength longer than 488 nm.
[0038]
In addition, when measuring a double-stained sample stained with two types of fluorescent dyes, the long-wavelength excitation light overlaps with the fluorescence from the short-wavelength excitation light. It is better to measure by removing the RF frequency corresponding to the wavelength. This is because the reflected light of the excitation light has a much higher intensity than the fluorescence wavelength.
[0039]
As described above, in the scanning laser microscope according to the first embodiment, the AOTF 311 is disposed on the fluorescent light path, and the frequency of the high-frequency voltage of the AOTF 311 is switched and set. Thereby, the fluorescence spectral characteristic data can be detected by detecting the amount of light passing through the AOTF 311 for each wavelength.
[0040]
Therefore, in the first embodiment, as described in FIG. 6 of Japanese Patent Laid-Open No. 2000-56244, which is a conventional method, “dispersing means such as a prism, and the dispersed fluorescence are condensed to obtain a spectrum sequence. A complex consisting of a condensing means for imaging and a digital mirror device (DMD) consisting of a group of electrically controllable micro-deflection mirrors arranged at the spectral line imaging position to select the wavelength range to be detected. It becomes possible to replace the method for obtaining the fluorescence spectral characteristics by the structure with one acousto-optic element. As a result, accurate spectral data with a simple configuration can be acquired at high speed by switching the frequency of the acoustooptic device, and miniaturization can be promoted. Further, as shown in FIG. 7 of Japanese Patent Laid-Open No. 2000-56244, it is possible to suppress optical loss due to fiber transmission that occurs in a configuration in which the spectroscopic unit is connected to the scanning device with a fiber in order to downsize the scanning device.
[0041]
Furthermore, according to the first embodiment, since the switching frequency of the RF frequency is 100 nsec to 1 μsec, spectral characteristic data can be acquired at high speed, so that rapid and highly accurate detection is realized. .
[0042]
In the scanning laser microscope according to the first embodiment, the acoustooptic device unit 31 is detachable from the scanning unit 20. Thereby, with the apparatus of only the fluorescence detection operation as a normal scanning laser microscope, and the apparatus of the present embodiment capable of selectively performing both normal fluorescence detection and spectral characteristic data of fluorescence, The common part of the scanning laser microscope can be shared. For this reason, it is possible to easily cope with system upgrade of the apparatus.
[0043]
In USP 5,377,003, USP 5,528, and 368, AOTF is used to perform monochromatic illumination or select observation light with a narrow bandwidth to perform fluorescence observation or Raman spectroscopy as in a CCD. A method that uses a two-dimensional detector is disclosed.
[0044]
In these, prisms with birefringence (mainly TiOF, which is the main part of AOTF). ₂ When a frequency is applied, internal distortion occurs in the prism itself. This internal distortion affects a configuration capable of two-dimensional detection such as a CCD. In accordance with the internal distortion of the frequency-applied prism, the image transmitted through the AOTF to be observed is deteriorated.
[0045]
In the present invention, in the following embodiments as well, two-dimensional detection like a CCD is not performed, but the amount of light emitted from one point of the sample is detected by scanning the sample surface. Therefore, in the present invention, since the amount of light is merely detected, such image deterioration does not occur.
[0046]
(Second Embodiment)
The second embodiment will be described with reference to FIG. FIG. 3 is a diagram showing a schematic configuration of a scanning laser microscope according to the second embodiment. In FIG. 3, the same parts as those in FIG. 1 are denoted by the same reference numerals, and the description thereof is omitted.
[0047]
In the second embodiment, the AOTF 311 has a function as the optical path switching mirror 32 by arranging the AOTF 311 at the arrangement position of the optical path switching mirror 32 described in FIG.
[0048]
In the above configuration, when a normal scanning image is acquired by the photodetector 34, the AOTF 311 is stopped without applying the RF frequency to the AOTF 311. As a result, all the fluorescence wavelengths are transmitted through the AOTF 311, guided to the photodetector 34 through the barrier filter 33, and detected.
[0049]
When acquiring the spectral characteristic data of fluorescence, the acoustooptic device 311 is applied with the frequency of the RF of the wavelength in substantially the same manner as in the first embodiment. As a result, the fluorescence wavelength is deflected by the AOTF 311 as described above, guided to the photodetector 312 and detected.
[0050]
According to the second embodiment, scanning is performed depending on whether light is detected by the photodetector 34 without applying any RF frequency to the AOTF 311 or detected by the photodetector 312 by applying the RF frequency. Images or spectral characteristic data can be acquired. Thereby, since the optical path switching mirror 32 can be saved as compared with the first embodiment, the configuration can be further simplified.
[0051]
In the first and second embodiments, the following modifications are possible. For example, in the first and second embodiments, spectral characteristic data is acquired by measuring the first-order diffracted light diffracted by the frequency of the RF with the photodetector 312, but transmitted through the acousto-optic element 311. The zero-order diffracted light to be detected may be detected by the photodetector 34. In this case, a light amount obtained by subtracting the light amount of the fluorescence wavelength (diffracted as the first-order diffracted light) corresponding to the RF frequency from the total light amount of the fluorescence wavelengths transmitted through the barrier filter 33 is detected for each RF frequency. It is detected by the device 312. By reversing these values, spectral characteristic data can be obtained. In this way, since the amount of fluorescent light is much larger than when detecting the first-order diffracted light, highly accurate data can be acquired.
[0052]
(Third embodiment)
FIG. 4 is a diagram showing a schematic configuration of a scanning laser microscope according to the third embodiment of the present invention. 4, the scanning laser microscope includes a light source unit 50, a scanning optical system 51, a microscope unit 52, a confocal optical system 53, an AOTF 311, detectors 312a and 312b, a control unit 40, and signal processing. A portion 45 is provided. 4, the same parts as those in FIG. 1 or FIG. 2 are denoted by the same reference numerals, and detailed description thereof is omitted.
[0053]
The difference between the scanning laser microscope shown in FIG. 4 and that in FIG. 1 is that the detectors 312a and 312b detect ± first-order diffracted light by the AOTF 311. For this reason, a signal processing unit 45 is newly provided.
[0054]
Light from the light source unit 50 is guided to the microscope unit 52 via the scanning optical system 51 as excitation light by the dichroic mirror 24 and excites the sample 27. The sample 27 labeled with a fluorescent reagent is excited by excitation light. The fluorescence emitted from the sample 27 is separated as fluorescence by the dichroic mirror 24 via the microscope unit 52 and the scanning optical system 51. Next, the light passes through the confocal optical system 53 and reaches the AOTF 311. Although the AOTF 311 is not illustrated, a specific driver for applying a specific frequency to the AOTF 311 is prepared in order to select a desired wavelength or wavelength width. Fluorescence is a wavelength component selected by the AOTF and polarized ± 1st order diffracted light which becomes polarized components orthogonal to each other, and 0th order diffracted light which becomes a component which is not further wavelength selected and is not deflected. It is separated into three optical paths.
[0055]
The ± first-order diffracted light is received by detectors 312a and 312b (hereinafter also referred to as “PMT”), the amount of light is detected, the signal processing unit 45 controls the signals from PMTs 312a and 312b, and data is obtained as sum signals or difference signals. Can be made.
[0056]
In this way, a detector is provided in each of the two fluorescent or reflected light paths deflected in different directions as wavelengths corresponding to the frequency applied by the acousto-optic device, and these detection signals are combined into a sum signal, a difference signal, or the like. If it is handled as a signal, it is possible to efficiently acquire the fluorescence of the desired wavelength or the total fluorescence intensity of the reflected light. Moreover, if it handles as an independent signal, the component derived from the sample can be acquired from the fluorescence or reflected light by which the desired wavelength was selected. These can be performed at high speed by an electric operation and can be easily switched.
[0057]
The operation of the AOTF 311 is performed as shown in FIG. 5 in combination with a driver not shown.
[0058]
First-order diffracted light having wavelength characteristics as shown in FIG. 5A can be obtained from the AOTF 311 according to the frequency applied from the driver. The frequency from the driver matches the wavelength λ 0, and a specific wavelength band is obtained as a spectrum based on the relationship between this frequency and the wavelength-dependent diffraction efficiency, and the wavelength resolution in the AOTF 311 can be determined. These usually differ depending on the characteristics of the light incident on the AOTF, such as the size, spread, wavelength, and polarization. However, since the scanning confocal laser microscope is used here, it is sufficient to handle a parallel fluorescent beam. A relatively high resolution of several nanometers to several tens of nanometers can be obtained. Further, according to the acoustooptic effect of AOTF 311, the ± first-order diffracted light includes substantially the same wavelength characteristics selected as described above, and is further separated into two polarization components orthogonal to each other in the positive and negative optical paths. , Respectively. The effect of this polarization separation is similar to that of a general polarization beam splitter. As a result, fluorescence that is randomly polarized light can be separated into two polarized light components in the same wavelength band, and the selected wavelength can be set freely.
[0059]
In addition, spectral detection is performed by setting a lower limit value λ1 and an upper limit value λ2 as shown in FIG. 5A for the wavelength, and sweeping the frequency corresponding to the band, that is, scanning the center wavelength. Can do. This operation does not require any mechanical operation and is performed by an electrical operation, so that it can be performed at a very high speed and without vibration. Furthermore, by combining with the scanning optical system 51 of the present embodiment, it becomes easy to achieve combined detection of two-dimensional and three-dimensional image acquisition and spectral acquisition at high speed.
[0060]
Further, the 0th-order diffracted light having a wavelength other than the selected wavelength passes through the birefringence crystal in the AOTF 311 and is trapped by an optical stopper that prevents stray light. At this time, the component contained in the 0th-order diffracted light is a fluorescence component other than the characteristics of FIG. 5 selected by the AOTF 311 among the fluorescence emitted from the sample 27, and the polarization characteristics of the fluorescence from the sample 27 are also retained. At this time, a slight light loss due to the transmittance in the crystal occurs in the 0th-order diffracted light. In general, fluorescence has a broad wavelength spectrum compared to the wavelength resolution of AOTF, and in addition, a plurality of fluorescent reagents may be used.
[0061]
On / off of specific wavelength selection on the AOTF 311 is easily achieved by electrical operation. Thereby, it is possible to perform spectroscopy according to the wavelength position and band to be detected. In other words, the AOTF 311 is electrically set so as not to be active (off) at the wavelength position (frequency) of the laser so that the leakage light of the laser beam having the excitation wavelength is not guided to the detection side, and the laser is completely cut. With this, spectral detection by wavelength selection and frequency sweep can be performed quickly. These can be easily achieved by electrically setting a plurality of wavelength positions (frequencies) on a driver even for a plurality of lasers.
[0062]
Further, a feature of the AOTF 311 is shown in FIG.
[0063]
The AOTF 311 has a characteristic of simultaneously applying a plurality of independent frequencies. FIG. 5B is an example in which three wavelength bands of λ0, λ3, and λ4 are obtained by giving three types of frequencies. The wavelength resolution for one frequency of the AOTF 311 is fixed at a narrow full width at half maximum (that is, high wavelength resolution) and cannot be varied in principle. Therefore, by bringing λ0, λ3, and λ4 closer to each other, specifically, by making the frequency applied to the AOTF 311 closer, the three spectra can form an envelope and the full width at half maximum can be expanded. It is also possible to set the lower limit value λ1 and the upper limit value λ2 as described above and sweep the envelope in the wavelength region. Thereby, it is possible to easily take a wider wavelength range and to deal with a sample emitting dark fluorescence. The number of frequencies that can be applied is determined by the combination of the characteristics of the AOTF 311 and the driver, and is not limited to the example described here, and more frequencies can be applied.
[0064]
As described above, by providing a plurality of frequencies to the acoustooptic device at the same time, a plurality of center wavelengths can be selected, and by bringing these center wavelengths close to each other, the relatively narrow wavelength resolution obtained by the conventional acoustooptic device can be further improved. A wide selection wavelength range can be set. Furthermore, by sweeping in the central wavelength region where a plurality of central wavelengths are set in the proximity of each other, efficient spectral data can be acquired even for a sample having a relatively dark fluorescence intensity. In this way, the method of setting a wider selection wavelength width by simultaneously applying a plurality of frequencies to the acoustooptic device is not limited to this embodiment, but other embodiments such as the first and second implementations. The present invention can be similarly applied to the embodiment and the following embodiments.
[0065]
Fluorescence has almost the same wavelength characteristics, but is separated into ± first-order diffracted light as polarized light components orthogonal to each other. The first-order diffracted lights are detected by the PMTs 312 a and 312 b, but at least two types of calculation signals can be obtained via the signal processing unit 45. If the signals from the PMTs 312a and 312b are output as sum signals from the signal processing unit 45, they can be handled as fluorescence total signals from the set wavelength width. Further, if signals from the PMTs 312a and 312b are output as a difference signal or an independent signal via the signal processing unit 45, they can be treated as a fluorescence polarization signal generated according to the actual state of the fluorescence reagent, and fluorescence polarization analysis can be performed. .
[0066]
FIG. 6 is a diagram showing a modification of the confocal optical system. FIG. 6A shows a confocal optical system in a common optical path which is raised as one embodiment of the present invention. On the other hand, since various kinds of light are usually diffracted in the spectral detection, generally only desired ± first-order diffracted light is detected as shown in FIG. 4, and in order to prevent stray light, a slit or a little is placed in front of the detectors 312a and 312b. Often places large pinholes. If these confocal optical systems and two slits are provided in the optical path, a light quantity loss due to vignetting is likely to occur. Therefore, in order to combine both functions and avoid loss, of course, a confocal optical system may be disposed in each of the ± first-order diffracted light paths diffracted in the optical system as shown in FIG. 6B. This increases the complexity of the apparatus, but it is also possible to have two effects of confocal effect and stray light cut.
[0067]
In the third embodiment, by using the AOTF 311 for spectral detection, there is no mechanical drive, high-efficiency spectroscopy can be performed, and furthermore, high-speed and high-resolution spectral detection (spectral data detection) can be performed. A microscope can be achieved. Furthermore, the polarization property of fluorescence can also be detected. By detecting the selected ± 1st order diffracted light (fluorescence component) with the same wavelength component and orthogonal polarization components, it is possible to easily switch to the same fluorescence sum signal and fluorescence polarization signal as usual, and generate in the actual sample. It becomes possible to quantify and examine the fluorescence in more detail.
[0068]
Although the conventional spectroscopic technique itself has a relatively large size of the apparatus, in this embodiment, the detector can be made relatively small by using AOTF.
[0069]
(Fourth embodiment)
FIG. 7 is a diagram showing a schematic configuration of a scanning laser microscope according to the fourth embodiment of the present invention.
[0070]
The scanning laser microscope according to the fourth embodiment has substantially the same configuration as that of the third embodiment. In the fourth embodiment, the detector side is modified in the third embodiment. That is, the third detector 312c is provided as a main part of the scanning laser microscope.
[0071]
In AOTF 311, the fluorescence is separated into ± 1st order diffracted light and 0th order diffracted light. The ± 1st-order diffracted light is as described above, but the 0th-order diffracted light is a component that is transmitted through the AOTF 311 and is not deflected, and has a component that is not wavelength-selected among the fluorescence from the sample 27. A third detector is provided in this optical path. As an example, a conventional filter type detector can be used. Specifically, a zero-order diffracted light after passing through AOTF 311 is provided with a detector having a barrier filter for extracting only desired fluorescence and a photomultiplier tube (PMT) by cutting the laser light, and the degree of stray light after AOTF 311 Depending on necessity, a confocal optical system composed of a confocal lens and a pinhole may be provided. The third detector 312c can be provided as a plurality of detectors as in the conventional scanning confocal laser microscope.
[0072]
That is, even if no special device is combined, the spectral detection can be performed by detecting ± first-order diffracted light using the AOTF 311 and the detection of wider width or the conventional detection can be performed using a filter. Can be easily switched. Here, in detection using a filter, it can be said that almost all methods that can be implemented with a scanning laser microscope using conventional filter detection can be used.
[0073]
Although not shown in the figure, a conventional filter type detector is given as an example of the third detector 312c. However, by using the optical path of the 0th-order diffracted light, other external spectrometers and avalanche photodiodes are used. Needless to say, it is possible to use (APD), a line sensor, a multi-channel sensor, and the like, and it can be more combined depending on the use range of this optical path.
[0074]
As described above, in addition to spectral detection by the AOTF 311, normal filter detection of components that are not polarized in the optical path of the 0th-order diffracted light is also possible. For example, a complex (hybrid) that can easily switch between spectral detection and filter-type detection. A scanning laser microscope can be used. Switching between the spectral detection and the conventional filter type detection is easy, and the spectral detection can easily contribute to the selection of a more optimal filter, so that a wide range of use can be covered depending on the purpose and usage.
[0075]
Thus, by arranging the third detector on the optical path of the 0th-order diffracted light that is not deflected by the frequency by the acoustooptic device, it is possible to detect the fluorescence or reflected light component that is not wavelength-selected, and to combine it as a device. It is possible to adopt a configuration capable of performing typical measurement.
[0076]
As described above, fluorescence or reflected light deflected in two different optical paths by the acoustooptic device is detected by at least one detector, and the desired center wavelength is selected by manipulating the frequency applied to the acoustooptic device. Alternatively, sweeping is performed in a desired wavelength region of the center wavelength, and desired fluorescence or reflected light can be efficiently acquired as spectral data at high speed and with high accuracy. In addition, in spite of this relatively simple configuration, the versatility is wide, the system can be configured with a small space, and the system can be downsized.
[0077]
(Fifth embodiment)
FIG. 8 is a diagram showing a schematic configuration of a scanning laser microscope according to the fifth embodiment of the present invention.
[0078]
The main part of the scanning laser microscope includes a light source unit 50 including a laser light source 22 and a collimator lens 21, a scanning optical system 51 including a galvano mirror 6 that performs two-dimensional scanning of XY, and a microscope including an imaging lens 8 and an objective lens 9. Of the unit 52, the confocal optical system unit 14 including the pinhole 13, the AOTF 311, which is an acoustooptic device, and the detectors 312a and 312b, which are two detectors, a photomultiplier tube (PMT), and the detectors 312a and 312b. It is comprised from the signal processing part 45 which controls a signal. Here, as in the first embodiment, the laser light source unit 50 may be introduced via an optical fiber. FIG. 8 shows a basic configuration of the system. In order to give the laser light source 22 a plurality of wavelengths, a multi-line laser or a plurality of laser light sources may be used.
[0079]
As the detector, a device that obtains an equivalent effect other than the PMT that can detect a signal through photoelectric conversion may be used.
[0080]
The operations and effects of the fifth embodiment are substantially the same as those of the third embodiment, but differ in the points described below.
[0081]
The laser light from the light source unit 50 passes through the AOTF 311 and the confocal optical system 53 and is guided as excitation light to the microscope unit 52 through the scanning optical system 51 to excite the sample 27. At this time, the confocal optical system 53 serves as one spatial filter for the excitation light. The sample 27 labeled with a fluorescent reagent is excited by excitation light. The fluorescence emitted thereby passes through the microscope unit 10 again, passes through the scanning optical system 51 and the confocal optical system 53, and reaches the AOTF 311. At this time, the confocal optical system functions to obtain a confocal effect in fluorescence. Although not shown, the AOTF 311 is provided with a specific driver that applies a specific frequency to the AOTF 311 in order to select a desired wavelength and wavelength width. The fluorescence emitted from the sample is separated into deflected ± first-order diffracted light which is a wavelength component selected by the AOTF 311 and becomes a polarization component orthogonal to each other.
[0082]
That is, the ATOF 15 has a function as a beam splitter that separates excitation light, which is laser light on the 0th-order diffracted light path, and ± 1st-order diffracted light obtained by selecting the wavelength of the fluorescence emitted from the sample 27 and deflecting it. The excitation light from the laser light source 22 is incident on the AOTF 311 from the direction opposite to the original traveling direction on the optical path of the 0th-order diffracted light of the AOTF 311. The wavelength characteristic of the dielectric film coated on the conventional dichroic mirror does not have such a steep rise characteristic, the maximum transmittance is a little low at 85%, the multi-band support is up to 3 excitation wavelengths, etc. There are drawbacks in coating technology. In the case of the fifth embodiment, since the AOTF 311 functions as a prism with respect to the excitation light and transmits the excitation light, the light amount loss basically occurs only in the transmittance of the crystal in the AOTF 311. In order to obtain higher efficiency, the loss can be reduced by rotating the polarization direction from the laser light source 22 with respect to the polarization direction of the birefringent crystal. At this time, if the laser beam is introduced into the AOTF 311 using a polarization-maintaining optical fiber, the alignment of the polarization direction can be achieved simply by rotating the direction of the fiber.
[0083]
For this reason, it is easy to handle multiple excitation wavelengths with high efficiency without having to activate the AOTF 311 electrically at those wavelength positions at the time of spectroscopic detection, without easily adding or changing the system. Can be done. Further, the wavelength selection of the ± first-order diffracted light can be performed with a high wavelength resolution unique to the AOTF 311. If it can be turned off in the vicinity of the position of the excitation wavelength as described above, the fluorescence can be spectrally acquired with a very high efficiency that has not been conventionally achieved. Of course, the confocal optical system 53 may be arranged as shown in FIG. 6B from the viewpoint of light utilization efficiency. Thus, by providing the AOTF 311 with the two functions of the beam splitter and the spectroscopic detection, a very simple configuration can be achieved and the system itself can be downsized.
[0084]
The non-deflected fluorescent component contained in the optical path of the 0th-order diffracted light can be separated using a dichroic mirror before reaching the laser light source, and a conventional filter type detector can be provided for complex measurement. is there. Needless to say, the AOTF 311 can be used to have the same functions and functions as those of the third and fourth embodiments.
[0085]
As described above, by providing the AOTF 311 with two functions of a beam splitter and a spectroscopic function, high-efficiency excitation light / fluorescence can be separated, and as a function of the AOTF 311 itself, high-speed and high-resolution spectroscopic detection can be achieved. It also becomes possible to reduce the size of the entire system. Furthermore, the polarization characteristics of ± 1st order diffracted light can be used to investigate the fluorescence polarization characteristics derived from the fluorescent reagent and the sample, and a more compact and more complex measurement can be performed.
[0086]
In this way, the laser light from the laser light source can be incident from the opposite direction to the traveling direction of the 0th-order diffracted light originally emitted from the acoustooptic element, and scanning is not affected by the frequency applied to the acoustooptic element. The sample can be irradiated with laser light through an optical system and a microscope. Accordingly, the acoustooptic device can function as a beam splitter described above with higher light utilization efficiency. In this case, the frequency applied to the acoustooptic device need not simply correspond to the known laser wavelength to be irradiated on the sample, and the separation of excitation light and fluorescence with high S / N that cannot be achieved with a conventional dichroic mirror. Can be achieved, and there is also a merit that it can cope with a plurality of laser wavelengths very easily.
[0087]
In addition to the efficient acquisition of spectral data using the acoustooptic device as described above, positive and negative first-order diffracted light, which are polarization components orthogonal to each other, are deflected in two different directions from the acoustooptic device, and at least one By detecting each with a detector, fluorescence or reflected light derived from a sample state or condition can be acquired as a polarization component.
[0088]
Therefore, the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the scope of the invention in the implementation stage. For example, in the present embodiment, the first-order diffracted light diffracted at the RF frequency is measured by the photodetector 312, and the spectral characteristic data is acquired. However, the zero-order diffracted light transmitted through the AOTF 311 is detected by the photodetector. 34 may be detected. In this case, a light amount obtained by subtracting the light amount of the fluorescence wavelength (diffracted as the first-order diffracted light) corresponding to the RF frequency from the total light amount of the fluorescence wavelengths transmitted through the barrier filter 33 is detected for each RF frequency. Detected by the instrument 312. By reversing these values, spectral characteristic data can be obtained. In this way, since the amount of fluorescent light is much larger than when detecting the first-order diffracted light, highly accurate data can be acquired. Further, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements.
[0089]
For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiment, the problems described in the column of problems to be solved by the invention can be solved, and the effects described in the effects of the invention can be obtained. In some cases, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0090]
As described in the third to fifth embodiments described above, if laser light using an optical fiber is introduced as a light source, as is generally known, avoidance of heat and vibration, a good point light source Downsizing of the scanning optical system and the detection system can be achieved.
[0091]
Further, as a light source, it is of course possible to combine a plurality of desired excitation wavelengths using a laser that generates a multiline or a combiner that combines beams using a plurality of lasers. It is also possible to provide a second AOTF there and to have functions of laser selection and intensity adjustment.
[0092]
Although not shown in the figure, ± first-order diffracted light is guided to one detector using a mirror or the like, and a shutter is provided in each optical path to detect or switch + 1st-order diffracted light and −1st-order diffracted light simultaneously. Alternatively, it may be detected alternately. If both shutters are opened at the same time, a total signal of fluorescence is obtained, and two different fluorescence polarization components can be detected by electrically synchronizing the shutter switching and detector timing and separating the signals temporally.
[0093]
In particular, the case where the reflected light is detected is not described in detail, but as an example, Raman occurs in the laser light irradiated on the sample 27, and the wavelength characteristic of the scattered light is examined according to the physical properties. The present invention can also be applied to spectroscopy. In this case, although the wavelengths of the laser light and the scattered light are very close to each other, Raman scattering can be efficiently detected by the high wavelength resolution by the AOTF 311 particularly by the configuration of the fifth embodiment. Thus, it goes without saying that the present invention is not necessarily limited to fluorescence spectroscopic detection and can be widely applied as a general spectroscope.
[0094]
Further, the present invention is described as a scanning confocal laser microscope, and the excitation light from the laser light source 22 is scanned on the sample surface 11 via the scanning optical system 7, but only one point is provided. Even in the basic configuration in which the excitation light is illuminated, the same operation and effect can be obtained by using the AOTF 311. In this case, in order to obtain desired data and image acquisition, a stage may be driven as scanning means, or other scanning means other than XY may be combined.
[0095]
The following invention is extracted from each of the above embodiments.
The scanning laser microscope according to the first aspect of the present invention includes an objective lens that focuses laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning and an acoustooptic that is arranged on the optical path of the fluorescent or reflected light and selectively deflects only light having a wavelength corresponding to the frequency of the high-frequency voltage applied from the incident fluorescent or reflected light And an optical detection unit that detects light passing through the acoustooptic device, and a frequency scanning unit that switches and sets a frequency of a high-frequency voltage applied to the acoustooptic device.
[0096]
Preferred embodiments of the scanning laser microscope according to the first aspect are as follows. Each of the following embodiments may be applied independently, or may be applied in appropriate combination.
[0097]
(1) A beam splitter that separates the laser light and the fluorescence or reflected light is further provided, and the beam splitter is disposed between the objective lens and the acoustooptic device.
[0098]
(2) The light detected by the light detection means is first-order diffracted light deflected by the acoustooptic device.
[0099]
(3) A second detector that detects zeroth-order light transmitted through the acoustooptic device is provided.
[0100]
(4) The frequency scanning means is switched and set so that the frequency characteristic of the high frequency voltage is synchronized with the scanning of the optical scanning means so that the spectral characteristics of each pixel can be detected.
[0101]
(5) The acoustooptic device and the light detection means are configured as one unit, and are detachably disposed in the detection light path separated by the beam splitter.
[0102]
(6) The scanning range by the frequency scanning means does not include the frequency of the high-frequency voltage corresponding to the laser wavelength.
[0103]
(7) The acoustooptic device deflects the fluorescence or the reflected light to first and second optical paths in different directions, and the detectors are disposed on the first and second optical paths, respectively. Including at least one detector.
[0104]
(8) A detector for detecting zero-order light transmitted through the acoustooptic device is further provided.
[0105]
(9) The acoustooptic device deflects the fluorescent light or the reflected light to the first and second optical paths in different directions and enters the laser light from a third optical path other than the first and second optical paths. To do.
[0106]
(10) The fluorescence or reflected light is positive or negative first-order diffracted light, which is polarized light components orthogonal to each other, on the first and second optical paths deflected by the acoustooptic device.
[0107]
(11) A signal processing unit that processes the signals from the two detectors provided in the deflected first and second optical paths as a sum signal or a difference signal, or as a separate signal, respectively. Prepared.
[0108]
(12) A plurality of center wavelengths or frequencies can be set in the acousto-optic device, and by making the plurality of center wavelengths or frequencies close to or adjacent to each other, a spectrum formed by each center wavelength or frequency is partially Superimpose and select a wider wavelength range.
[0109]
(13) The acoustooptic device is configured to polarize the fluorescence or the reflected light into first and second optical paths in different directions, and the fluorescence guided to the first and second optical paths or the A deflecting device that guides the reflected light to one detector is provided.
[0110]
(14) The apparatus further includes a shutter provided in each of the first and second optical paths.
[0111]
(15) The third optical path is an optical path of zero-order diffracted light that is not deflected at the frequency of the acoustic element.
[0112]
(16) The laser beam on the third optical path is on the optical path of the 0th-order diffracted light emitted from the acoustooptic device, and the traveling direction thereof is opposite to the 0th-order diffracted light.
[0113]
A scanning laser microscope according to a second aspect of the present invention includes an objective lens that collects laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning, an acoustooptic element arranged on the optical path of the fluorescence or reflected light, and the acoustooptic element, when a high frequency voltage is applied, the high frequency of the incident fluorescence or reflected light Light having a wavelength corresponding to the frequency of the voltage is diffracted into + 1st order diffracted light and −1st order diffracted light, and when the high frequency voltage is not applied, the incident fluorescence or reflected light is transmitted as 0th order diffracted light, and the acoustooptic A high-frequency signal control unit that controls on / off of application of the high-frequency voltage to the element and sets the high-frequency voltage, a first photodetector that receives the + 1st-order diffracted light, and the -1st-order diffraction Characterized by comprising a second photodetector for receiving the.
[0114]
Preferred embodiments of the scanning laser microscope according to the second aspect are as follows. Each of the following embodiments may be applied independently, or may be applied in appropriate combination.
[0115]
(1) The apparatus further includes means for generating an image of the sample based on a value obtained by adding the output signal of the first photodetector and the output signal of the second photodetector.
[0116]
(2) The spectrum of the fluorescence or reflected light is acquired by sweeping the frequency of the high-frequency voltage by the high-frequency signal controller and acquiring the output signals of the first and second photodetectors at each frequency. To do.
[0117]
(3) In (2), further comprising a third photodetector for receiving the 0th-order diffracted light, and when the high-frequency voltage is not applied to the acoustooptic device, the output signal of the third photodetector is To generate an image of the sample.
[0118]
A scanning laser microscope according to a third aspect of the present invention includes an objective lens that focuses laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning, an acoustooptic element arranged on the optical path of the fluorescence or reflected light, and the acoustooptic element, when a high frequency voltage is applied, the high frequency of the incident fluorescence or reflected light Light having a wavelength corresponding to the frequency of the voltage is diffracted into + 1st order diffracted light and −1st order diffracted light, and when the high frequency voltage is not applied, the incident fluorescence or reflected light is transmitted as 0th order diffracted light, and the acoustooptic A high-frequency signal control unit that controls on / off of application of the high-frequency voltage to the element and sets the high-frequency voltage, a first photodetector that receives the + 1st-order diffracted light, and the -1st-order diffraction A second photodetector for receiving the light, and the laser light source transmits the acoustooptic element without being modulated and guided to the objective lens so as to be guided to the objective lens. It is arranged in the optical path.
[0119]
A scanning laser microscope according to a fourth aspect of the present invention includes an objective lens that condenses laser light on a sample and captures fluorescence or reflected light from the sample, and two-dimensionally the laser light on the sample. Optical scanning means for scanning, an acoustooptic element arranged on the optical path of the fluorescence or reflected light, and the acoustooptic element, when a high frequency voltage is applied, the high frequency of the incident fluorescence or reflected light While diffracting light having a wavelength corresponding to the frequency of the voltage into first-order diffracted light and not applying the high-frequency voltage, the incident fluorescence or reflected light is transmitted as zero-order diffracted light, and the high-frequency voltage applied to the acoustooptic device A high-frequency signal control unit that controls the on / off of the application of light and sets the high-frequency voltage; and a photodetector that receives the first-order diffracted light. Sweeping the frequency of the high-frequency voltage, by obtaining the output signal of the photodetector at each frequency, to obtain the spectral data of the fluorescence or the reflected light.
[0120]
4th aspect WHEREIN: The 2nd photodetector which light-receives the said 0th-order diffracted light is further provided, When the said high frequency voltage is not applied to the said acoustooptic device, it uses the output signal of the said 2nd photodetector. Preferably, an image of the sample is generated.
[0121]
The present invention is not limited to the above-described embodiments. Of course, various modifications can be made without departing from the scope of the present invention.
[0122]
【The invention's effect】
According to the present invention, it is possible to provide a scanning laser microscope that can be simplified in configuration, promote miniaturization, can perform high-efficiency spectroscopy, and can also realize high-speed and high-resolution spectral detection. it can.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing a main part of a scanning laser microscope according to a first embodiment of the present invention.
2 is a characteristic diagram showing an example of spectral characteristic data acquired in FIG. 1. FIG.
FIG. 3 is a configuration diagram showing a main part of a scanning laser microscope according to a second embodiment of the present invention.
FIG. 4 is a diagram showing a schematic configuration of a scanning laser microscope according to a third embodiment.
FIG. 5 is a diagram for explaining an example of increasing the bandwidth in a pseudo manner.
FIG. 6 is a diagram showing a modification of the confocal optical system.
FIG. 7 is a diagram showing a schematic configuration of a scanning laser microscope according to a fourth embodiment.
FIG. 8 is a diagram showing a schematic configuration of a scanning laser microscope according to a fifth embodiment.
[Explanation of symbols]
1. Acousto-optic element
6 ... Galvano mirror
7. Scanning optical system
8 ... Imaging lens
9 ... Objective lens
10. Microscope part
11 ... Sample surface
13 ... pinhole
14 ... Confocal optical system
15 ... ATOF
19 ... Reflecting mirror
20 ... Scanning unit
21 ... Collimator lens
22 ... Laser light source
23 ... Fiber
24 ... Dichroic mirror (beam splitter)
25. Optical scanning means
26 ... Objective lens
27 ... Sample
28 ... Confocal lens
29 ... Confocal pinhole (confocal spot)
30 ... Lens
31 ... Acousto-optic element unit
32 ... Mirror
32a ... position
32b ... position
33 ... Barrier filter
34. Photodetector
40 ... Control unit

Claims (25)

An objective lens that focuses laser light on the sample and captures fluorescence from the sample;
Optical scanning means for two-dimensionally scanning the laser beam on the sample;
Wherein arranged fluorescence light path, and an acoustic optical element which only light having a wavelength corresponding to the frequency of the high frequency voltage selected by deflection applied from the fluorescence incident,
Photodetection means for detecting fluorescence deflected by the acoustooptic device;
A scanning laser microscope comprising: frequency scanning means for switching and setting a frequency of a high-frequency voltage applied to the acoustooptic device. 2. The scanning laser microscope according to claim 1, further comprising a beam splitter for separating the laser beam and the fluorescence, and the beam splitter is disposed between the objective lens and the acoustooptic device.   3. The scanning laser microscope according to claim 1, wherein the light detected by the light detection means is first-order diffracted light deflected by the acoustooptic device.   The scanning laser microscope according to claim 3, further comprising a second detector that detects zeroth-order light transmitted through the acoustooptic device.   5. The scanning laser microscope according to claim 1, wherein the frequency scanning unit detects a spectral characteristic for each pixel by synchronizing a frequency of a high-frequency voltage with scanning of the optical scanning unit. Change settings to obtain.   6. The scanning laser microscope according to claim 2, wherein the acoustooptic device and the light detection unit are configured as one unit, and are detachably attached to a detection light path separated by the beam splitter. It arranges in.   7. The scanning laser microscope according to claim 1, wherein a scanning range by the frequency scanning unit does not include a frequency of a high-frequency voltage corresponding to a laser wavelength. The scanning laser microscope according to claim 1 or 2,
The acousto-optic device deflects the fluorescence into first and second optical paths in different directions;
The detector includes at least one detector disposed on each of the first and second optical paths.   9. The scanning laser microscope according to claim 8, further comprising a detector for detecting 0th-order light transmitted through the acoustooptic device. 9. The scanning laser microscope according to claim 1, wherein the acoustooptic device deflects the fluorescence into first and second optical paths in different directions and other than the first and second optical paths. Laser light is incident from the third optical path. 11. The scanning laser microscope according to claim 8 , wherein the fluorescence on the first and second optical paths deflected by the acousto-optic element is a positive or negative polarization component that is a polarization component orthogonal to each other. First-order diffracted light.   12. The scanning laser microscope according to claim 8, wherein a signal from two detectors provided in the deflected first and second optical paths is a sum signal or a difference signal. And a signal processing unit for processing each of the combined signals or independent signals.   The scanning laser microscope according to any one of claims 1, 2, 3, 4, and 8 to 11, wherein the acoustooptic device has a plurality of center wavelengths or frequencies. By setting the plurality of center wavelengths or frequencies close to or adjacent to each other, spectra formed by the respective center wavelengths or frequencies are partially overlapped so that a wider wavelength width can be selected. The scanning laser microscope according to claim 11,
The acoustooptic device deflects the fluorescence into first and second optical paths in different directions,
A deflecting device is provided for guiding the fluorescence guided to the first and second optical paths to one detector.   15. The scanning laser microscope according to claim 14, further comprising a shutter provided in each of the first and second optical paths.   11. The scanning laser microscope according to claim 9, wherein the third optical path is an optical path of zero-order diffracted light that is not deflected at the frequency of the acoustic element.   11. The scanning laser microscope according to claim 10, wherein the laser light on the third optical path is on an optical path of zero-order diffracted light emitted from the acoustooptic device, and the traveling direction thereof is the same as that of the zero-order diffracted light. Is the opposite. An objective lens that focuses laser light on the sample and captures fluorescence from the sample;
Optical scanning means for two-dimensionally scanning the laser beam on the sample;
The acoustooptic element disposed on the fluorescence optical path, and the acoustooptic element, when a high frequency voltage is applied, light of a wavelength corresponding to the frequency of the high frequency voltage of the incident fluorescence is converted to + 1st order diffracted light. Diffracted into −1st order diffracted light, and when the high frequency voltage is not applied, transmits the incident fluorescence as 0th order diffracted light,
A high-frequency signal control unit configured to control the on / off of the application of the high-frequency voltage to the acoustooptic device and to set the high-frequency voltage;
A first photodetector for receiving the + 1st order diffracted light;
A scanning laser microscope comprising: a second photodetector that receives the −1st order diffracted light.   19. The scanning laser microscope according to claim 18, further comprising means for generating an image of the sample based on a value obtained by adding the output signal of the first photodetector and the output signal of the second photodetector. It has. The scanning laser microscope according to claim 18, wherein the high-frequency signal control unit sweeps the frequency of the high-frequency voltage to obtain output signals of the first and second photodetectors at each frequency. Obtain fluorescence spectral data. The scanning laser microscope according to claim 20,
A third photodetector for receiving the zero-order diffracted light;
When the high-frequency voltage is not applied to the acoustooptic device, an image of the sample is generated using the output signal of the third photodetector. An objective lens that focuses laser light on the sample and captures fluorescence from the sample;
Optical scanning means for two-dimensionally scanning the laser beam on the sample;
The acoustooptic element disposed on the fluorescence optical path, and the acoustooptic element, when a high frequency voltage is applied, light of a wavelength corresponding to the frequency of the high frequency voltage of the incident fluorescence is converted to + 1st order diffracted light. Diffracted into −1st order diffracted light, and when the high frequency voltage is not applied, transmits the incident fluorescence as 0th order diffracted light,
A high-frequency signal control unit configured to control the on / off of the application of the high-frequency voltage to the acoustooptic device and to set the high-frequency voltage;
A first photodetector for receiving the + 1st order diffracted light;
A second photodetector that receives the −1st order diffracted light, and the laser light source transmits the acoustooptic element without being modulated and is guided to the objective lens. A scanning laser microscope disposed in the optical path of the 0th-order diffracted light. An objective lens that focuses laser light on the sample and captures fluorescence from the sample;
Optical scanning means for two-dimensionally scanning the laser beam on the sample;
An acoustic optical element disposed on an optical path of the fluorescence, the acoustooptic element, when a high frequency voltage is applied, light having a wavelength corresponding to the frequency of the high frequency voltage of the fluorescence incident 1-order diffracted light When diffracting and when the high-frequency voltage is not applied, the incident fluorescence is transmitted as zero-order diffracted light,
A high-frequency signal control unit configured to control the on / off of the application of the high-frequency voltage to the acoustooptic device and to set the high-frequency voltage;
A photodetector that receives the first-order diffracted light, sweeps the frequency of the high-frequency voltage by the high-frequency signal control unit, and acquires the output signal of the photodetector at each frequency, thereby obtaining the fluorescence A scanning laser microscope that acquires spectral data. The scanning laser microscope according to claim 23,
A second photodetector for receiving the zeroth-order diffracted light;
When the high-frequency voltage is not applied to the acoustooptic device, an image of the sample is generated using the output signal of the second photodetector. 24. The scanning laser microscope according to claim 18, further comprising a beam splitter that separates the laser light and the fluorescence, and the beam splitter is disposed between the objective lens and the acoustooptic device. ing.

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