JP2013080024A - Scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a scanning microscope that can obtain an image of a specimen beyond an optical limit of an optical system.SOLUTION: A scanning microscope 10 includes: an illumination optical system that scans a specimen 70 with illumination light radiated from a light source device 20 using a scanning optical system 30; a light collecting optical system that guides the light from the specimen 70 to a detecting unit (for example, first detecting unit 40); and a control unit 100 that controls a scanning position of the illumination light on the specimen 70 by the scanning optical system 30, and controls the intensity of the illumination light to change to be the 1/n-th power of a sine waveform in accordance with the scanning position. Here, n is an integer equal to or greater than 1.

Description

  The present invention relates to a scanning microscope.

  Conventionally, various techniques have been proposed for super-resolution techniques for observing a specimen with a resolution higher than that of a microscope optical system. Among them, there is a method of combining structured illumination and image processing (see, for example, Patent Document 1).

JP 2008-216778 A

  As a method of performing structured illumination, for example, there is a configuration in which the sample is irradiated with illumination light spatially modulated by a diffraction grating, but a configuration is made so that a conjugate plane of the sample is formed in a common optical path of excitation light and fluorescence. It is not realistic to apply to a general scanning microscope.

  The present invention has been made in view of such problems, and an object of the present invention is to provide a scanning microscope that can acquire an image of a specimen that exceeds the optical limit ability of an optical system.

  In order to solve the above-described problems, a scanning microscope according to the present invention includes an illumination optical system that scans a specimen using a scanning unit with illumination light emitted from a light source unit, and a light guide that guides light from the specimen to a detection unit. A control unit for controlling the scanning position of the illumination light on the sample by the scanning unit and controlling according to the scanning position so that the intensity change of the illumination light becomes a 1 / nth power of a sinusoidal waveform; It is characterized by having. However, n is an integer of 1 or more.

  In such a scanning microscope, the light source unit includes an acoustooptic device, and the control unit controls the acoustooptic device using a 1 / nth power drive signal of a sinusoidal waveform according to the scanning position, It is preferable to control so that the intensity change of the illumination light becomes a 1 / nth power of a sinusoidal waveform.

  Further, in such a scanning microscope, the light source unit includes a laser light source, and the control unit controls the laser light source using a 1 / nth power drive signal of a sinusoidal waveform according to the scanning position, It is preferable to control so that the intensity change of the illumination light becomes a 1 / nth power of a sinusoidal waveform.

  In such a scanning microscope, it is preferable that the control unit generates an image of the sample by calculation from a plurality of images acquired in an illumination state in which the phase of the sinusoidal waveform of the illumination light is changed.

  In such a scanning microscope, a plurality of images acquired in an illumination state in which the phase of the sinusoidal waveform of the illumination light is changed can be obtained by changing the phase of the illumination fringe pattern by changing the phase of the sinusoidal waveform of the illumination light. It is preferable that they are a plurality of images acquired in an illumination state in which is changed.

  In such a scanning microscope, a plurality of images acquired in an illumination state in which the phase of the sinusoidal waveform of the illumination light is changed are illuminated by changing the phase of the sinusoidal waveform of the illumination light for each scanning line. It is preferable that the images are a plurality of images acquired in an illumination state in which the stripe pattern is rotated.

  In such a scanning microscope, the control unit circulates and reads out data using a drive waveform table storing data for one period of intensity modulation and a position designated as a preset value of the drive waveform table as a start position. It is preferable to control the sinusoidal waveform by data read out by setting a preset value according to the illumination state.

  In addition, such a scanning microscope preferably further includes a detection unit that is disposed at a position facing the illumination optical system via the sample and detects illumination light transmitted through the sample.

  When the present invention is configured as described above, it is possible to provide a scanning microscope that can acquire an image of a specimen that exceeds the optical limit capability of the optical system.

It is explanatory drawing which shows the structure of a scanning microscope. It is explanatory drawing for demonstrating the illumination state for acquiring the image of a super-resolution. It is explanatory drawing which shows the structure of the control part of the said scanning microscope. It is explanatory drawing which shows the structure of an illumination fringe signal generator. It is explanatory drawing which shows the timing of the synchronizing signal output from a synchronizing signal generator. It is explanatory drawing for demonstrating the drive method of AOTF, (a) shows a drive waveform, (b) shows the structure of a drive waveform table. It is explanatory drawing which shows the drive waveform of the striped illumination which shifted the phase. It is explanatory drawing which shows the drive waveform in the case of carrying out pi / 3 rotation of fringe illumination. It is explanatory drawing which shows the drive waveform for two-photon excitation.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of the optical system of the scanning microscope will be described with reference to FIG. This scanning microscope 10 includes a scanning optical system 30 that scans a sample 70 placed on a stage by irradiating laser light (illumination light) emitted from a light source device 20, and fluorescence (observation light) from the sample 70. ), And a third detector 60 that detects illumination light transmitted through the specimen 70. In the following description, the optical axis direction of the scanning optical system 30 is defined as the z axis, and the directions orthogonal to each other in a plane orthogonal to the z axis are defined as the x axis and the y axis, respectively.

  In this scanning microscope 10, the light source device 20 includes a light source 21 configured to be able to emit a plurality of laser beams having different wavelengths simultaneously or individually, and a laser beam that passes under the control of an AOTF driver described later. Is composed of an AOTF (acousto-optic element) 22 which is an intensity modulation element that changes (intensity modulates) the intensity between 0 and 100%. In the following description, it is assumed that two types of laser light, infrared light and visible light, are emitted from the light source 21. The infrared light emitted from the light source 21 is a very short pulsed light (for example, 100 femtosecond pulsed light) emitted at a predetermined period for inducing multiphoton excitation of the specimen 70. And hereinafter referred to as “IR pulsed light”).

  The scanning optical system 30 includes a first dichroic mirror 31, a scanning unit 32, a scan lens 33, a second objective lens 34, a second dichroic mirror 35, and an objective lens 36 in order from the light source device 20 side. Has been.

  The first detection unit 40 is an NDD (Non Descanned Detector), and is disposed on the side of the second dichroic mirror 35. The first condensing lens 41, the second condensing lens 41 are arranged in this order from the second dichroic mirror 35 side. The condenser lens 42 and the first photodetector 43 are included. Here, the light receiving surface of the first photodetector 43 is arranged substantially conjugate with the position of the exit pupil P of the objective lens 36.

  The second detection unit 50 is disposed on the side of the first dichroic mirror 31, and in order from the first dichroic mirror 31 side, the third condenser lens 51 and the focal plane on the sample side of the objective lens 36. The light shielding plate 52 and the second photodetector 53 are arranged at substantially conjugate positions. Here, the light shielding plate 52 is provided with a pinhole 52 a, and the pinhole 52 a is disposed so as to include the optical axis of the scanning optical system 30.

  The third detection unit 60 is disposed on the opposite side of the objective lens 36 with the sample 70 interposed therebetween, and includes a fourth condenser lens 61 and a third photodetector 62 in order from the sample 70 side. Yes.

  In addition, the scanning microscope 10 controls the operation of the AOTF 22 and the scanning unit 32, the position where the scanning unit 32 scans the laser beam (coordinates within the scanning plane on the specimen 70), the first, second, and A control unit 100 (not shown in FIG. 1) that processes values detected by the third photodetectors 43, 53, and 62 is provided. As the first to third photodetectors 43, 53, and 62, for example, a PMT (Photo Multiplier Tube) is used.

  In the scanning microscope 10, the laser light, which is a substantially parallel light beam emitted from the light source 21 of the light source device 20, passes through the AOTF 22, passes through the first dichroic mirror 31, and enters the scanning unit 32. The scanning unit 32 scans the laser light two-dimensionally in the direction orthogonal to the optical axis (the above-described x-axis direction and y-axis direction). For example, the scanning unit 32 reflects the laser light to reflect the laser light. A first deflection element that deflects in a predetermined direction (this direction is defined as an x-axis direction) in a plane orthogonal to the optical axis, and a deflection direction of the first deflection element in a plane orthogonal to the optical axis. It consists of two deflecting elements comprising a second deflecting element that deflects in an orthogonal direction (this direction is the y-axis direction). The laser light (substantially parallel light beam) emitted from the scanning unit 32 is imaged on the primary image plane I by the scan lens 33 and then passes through the second objective lens 34 to become a substantially parallel light beam again. The light passes through the dichroic mirror 35 and is collected by the objective lens 36 on the focal plane of the objective lens 36 on the specimen 70. The laser beam condensed on the specimen 70 is a point image, and the diameter of the point image is determined by the numerical aperture (NA) of the objective lens 36.

  Here, in the scanning microscope 10 according to the present embodiment, at the time of observing the sample 70, either the IR pulse light or the visible light among the laser light emitted from the light source 21 obtains an image of the sample 70. It is used as illumination light (excitation light) for imaging.

  For example, when IR pulse light is used as illumination light (excitation light), visible light is not used at all, or is used for light stimulation of the specimen 70. In this case, when the sample 70 is irradiated with IR pulse light, fluorescence by multiphoton excitation is generated from the sample 70, and this fluorescence becomes observation light and enters the objective lens 36, and is substantially parallel by the objective lens 36. It becomes a light beam, is reflected by the second dichroic mirror 35, and enters the first detection unit 40. The observation light is converted into a substantially parallel light beam having a required diameter by the first condenser lens 41 and the second condenser lens 42, and is incident on the first photodetector 43 and detected. In addition, in this 1st detection part 40, in order to remove IR pulse light which is reflected by the sample 70, the objective lens 36, etc., and injects into this 1st detection part 40 with observation light, IR cut filter which is not illustrated is arrange | positioned. You may do it.

  On the other hand, when using visible light as illumination light (excitation light), IR pulse light may be used for light stimulation of the specimen 70. In this case, when the observation light is irradiated onto the specimen 70, fluorescence is generated from the specimen 70, and this fluorescence becomes observation light and enters the objective lens 36. The objective lens 36 becomes a substantially parallel light beam, and the second dichroic. The light passes through the mirror 35. Then, this observation light is imaged on the primary image plane I by the second objective lens 34, further converted into a substantially parallel light beam by the scan lens 33, and incident on the scanning unit 32, and descanned by the scanning unit 32. The light is emitted, reflected by the first dichroic mirror 31, enters the second detection unit 50, and is condensed on the pinhole 52 a of the light shielding plate 52 by the third condenser lens 51. In the second detector 50, only the light that has passed through the pinhole 52 a of the light shielding plate 52 reaches the second photodetector 53 and is detected.

  As described above, the pinhole 52a of the light shielding plate 52 is conjugate with the point image of the laser beam condensed on the scanning surface on the specimen 70, and exits from the irradiation area on the specimen 70 (focal plane of the objective lens 36). The observed light (fluorescence) can pass through the pinhole 52a. On the other hand, most of the light emitted from other regions on the specimen 70 is not condensed on the pinhole 52a and cannot pass therethrough.

  As described above, the scanning optical system 30 includes the illumination optical system and the scanning unit that scan the sample 70 with the illumination light emitted from the light source device 20 that is the light source unit, and the light from the sample 70 as the first and second detection units. 40 and 50 have a function of light collecting means. Then, the control unit 100 (not shown) processes the optical signals detected by the first and second photodetectors 43 and 53 in synchronization with the scanning of the scanning unit 32, so that the scanning position of the laser light on the specimen 70 is determined. A two-dimensional image on the scanning surface of the specimen 70 can be obtained using the luminance obtained from the optical signal. As a result, the scanning microscope 10 can obtain an image of the specimen 70 with high resolution. Thus, the scanning microscope 10 according to the present embodiment can be used as both a scanning multiphoton microscope and a scanning confocal microscope. In addition, when using as a scanning multiphoton microscope, the 2nd detection part 50 is unnecessary, and when using as a scanning confocal microscope, the 1st detection part 40 is unnecessary.

  Here, the scanning microscope 10 according to the present embodiment is provided in the light source device 20 in order to obtain an image with a resolution exceeding the optical resolution of the scanning microscope 10 (hereinafter referred to as “super-resolution”). The laser beam transmittance of the AOTF 22 is changed according to the operation of the scanning unit 32. At this time, when IR pulse light is used as excitation light (two-photon excitation), the square of the intensity change becomes a sine wave shape with respect to the scanning region on the specimen 70 (the intensity change of the laser light is 1 / sine of the sine wave). In the case where the intensity of the laser light is modulated so that it becomes square (2) and visible light is used as the excitation light (one-photon excitation), the laser has a sinusoidal intensity change with respect to the scanning region on the specimen 70. A plurality of images with different illumination states are acquired by changing the intensity of light. That is, it is performed so that the nth power of the intensity change of the laser light becomes a sine wave shape, that is, the intensity change of the laser light becomes 1 / nth power of the sine wave waveform (n is a photon of the excitation light at the time of excitation) number). Specifically, when visible light is used as excitation light (one-photon excitation), as shown in FIG. 2, a striped illumination state in which the intensity of the laser light changes sinusoidally in the x-axis direction as shown in FIG. A reference (this illumination state is referred to as “0 phase”), and the illumination state in which the phase of the sinusoidal waveform is shifted from the 0 phase state by −2π / 3 and + 2π / 3 in the x-axis direction (“−2π / 3 respectively”). The three illumination states (referred to as “phase” and “+ 2π / 3 phase”) are set as one set (this set is referred to as “θrad”). Rotate by + π / 3 rad and −π / 3 rad to the center (each set is called “θ + π / 3 rad” and “θ-π / 3 rad”), and acquire images of the sample 70 in a total of nine illumination states (9 sheets) Images) and super-resolution images from these images Is configured to get.

  Hereinafter, a configuration for scanning the specimen 70 by modulating the intensity of laser light will be described with reference to FIG. FIG. 3 mainly shows the configuration of the control unit 100 of the scanning microscope 10, and only the main part of the optical system is shown. Further, here, a case will be described in which visible light is used as excitation light (one-photon excitation), and fluorescence emitted from the specimen 70 is used to acquire an image using the second detection unit 50.

  The control unit 100 synchronizes the transmittance of the AOTF 22, the scanning position (coordinates) of the laser light (illumination light) by the scanning unit 32, and the detection and storage of fluorescence (observation light) by the second photodetector 53. A synchronizing signal generator 101 for generating a synchronizing signal, an illumination fringe signal generator 102 for generating a signal (illumination fringe signal) for modulating the intensity of the laser light by the synchronizing signal, and an analog signal for the illumination fringe signal which is a digital signal An external DA conversion element 103 for conversion to A, an AOTF driver 104 for controlling the transmittance of the AOTF 22 by an analog illumination fringe signal, and a first and second deflection of the scanning unit 32 by a drive signal output from the synchronization signal generator 101. A scanner driver 105 for controlling the rotation angle of the element; a signal amplifier 106 for amplifying the output signal of the second photodetector 53; and a synchronization signal The signal output from the raw device 101, AD converter 107 for converting an output signal of the second photodetector 53 (analog signal) into a digital signal and,, and a memory 108 for storing the digital signal.

  Here, a case where an image (point image) of 512 × 512 points on the scanning region is acquired will be described as an example. In the following description, the synchronization signal generator 101 receives the reference clock clkA for driving the AOTF 22, the pixel clock pclk indicating the timing for acquiring the point image on the scanning region, the horizontal synchronization signal Hsync, and the point image information. The memory control mem_cnt indicating the timing to be stored in the memory 108 and the drive signal sigA for operating the scanning unit 32 are output.

  In the synchronization signal generator 101, the pixel clock pclk is an equally-spaced clock divided by one line 512, and generates a constant-speed drive signal sigA in synchronization therewith. The horizontal synchronization signal Hsync is a signal synchronized in the horizontal direction for each line (a clock that changes every time the pixel clock pclk is counted 512). Further, the synchronization signal generator 101 outputs the reference clock clkA synchronized with the pixel clock pclk to the illumination fringe signal generator 102, and the illumination fringe signal generator 102 uses the reference clock clkA synchronized with the pixel clock pclk. The signal can be configured. FIG. 5 shows a schematic timing chart of the drive signal sigA, the horizontal synchronization signal Hsync, the pixel clock pclk, and the reference clock clkA synchronized with the pixel clock pclk.

  Next, a method of generating intensity modulation of the laser light necessary for acquiring nine images and the illumination fringe signal generator 102 will be described. Now, as shown in FIG. 6A, a sine wave drive waveform for generating illumination fringes is divided into 300 discrete values, each of which is designated as N0 to N299, and this data is referred to as “drive data”. . When the drive waveform shown in FIG. 6A is 0 phase in the above-described θrad, these drive data are held in the drive waveform table 102b shown in FIG. 4, and the reference clock clkA generated by the synchronization signal generator 101 is used. 300 data is counted as one cycle by the counter 102a, and the data is sequentially read from the drive waveform table 102b. In the zero phase, the preset value is zero, and the counter 102a is reset in one cycle. For the waveforms shifted by + 2π / 3 phase and −2π / 3 phase, the preset values may be 100 and 200. That is, when the phase is + 2π / 3, the drive data may be read from N100, and when the phase is −2π / 3, the drive data is read from N200 and the drive waveform is shifted as shown in FIG. be able to. As described above, when data is read up to N299, the counter 102b is reset and data is sequentially read from N0 (this operation is referred to as “circular read”).

  Here, assuming that the resolution limit of the objective lens 36 is fmax, the resolution limit is defined by fmax = 2NA / λ from the numerical aperture NA of the objective lens 36 and the wavelength λ. On the other hand, if the sampling spatial frequency is fsample, this spatial frequency is fsample = 2 / P from the pixel size P for obtaining the sample image. In order for the image acquired by the scanning microscope 10 to exceed the resolution limit of the objective lens 36, it is necessary to have a relationship of fmax <fsample. In real space, the resolution limit of the objective lens 36 is 2.5. It is desirable to have about 3 pixels. That is, in the case of less than 2 pixels, it can be said that the performance of the objective lens 36 is not utilized. For this reason, the above-described driving waveform needs to be one cycle with two or more pixels. In the present embodiment, the drive waveform is controlled to be one cycle with three pixels (pixel clock pclk is three clocks).

  Next, a method for rotating the illumination stripe will be described. In this case as well, a preset value for reading drive data from the drive waveform table 102b by the illumination fringe signal generator 102 may be set in accordance with each rotation. However, if only the phase is shifted, it is applied to all lines. While it is sufficient to give the same preset value for one image, it is necessary to set the preset value for each line in the case of rotation. For example, in the case of rotating π / 3 rad from 0 phase (when 0 phase of θ + π / 3 rad is set), the drive waveform may be shifted by π / 3 phase for each line, so the preset value is set to 0 for the first line. The second line has a preset value of 50, the third line has a preset value of 100, the fourth line has a preset value of 150, the fifth line has a preset value of 200, and the sixth line has a preset value of 250. To do. Assuming one cycle from the 1st line to the 6th line, the preset value for the 7th line is set to 0 again, and if the preset value is set by repeating the same, the illumination fringe pattern rotated by + π / 3 rad as shown in FIG. Can be generated. Similarly, nine types of illumination fringe patterns as shown in FIG. 2 can be constructed by appropriately giving preset values to other images as well.

  The illumination fringe signal generator 102 generates an illumination fringe signal (illumination pattern), and the external DA converter 103 inputs an analog signal (sine wave drive signal) synchronized with the reference clock clkA to the AOTF driver 104. The intensity of the laser beam is modulated by controlling the transmittance. The fluorescence obtained by excitation with the laser light is converted into an electric signal by the second photodetector 53, then amplified by the signal amplifier 106, and sampled in synchronization with the pixel clock pclk by the AD conversion element 107. Is done. The image data sampled by the AD conversion element 107 is controlled by a memory control signal mem_cnt generated by the synchronization signal generator 101 and stored in the memory 108.

  As described above, in the scanning microscope 10 according to the present embodiment, the drive waveform for one period of 0 phase at θ rad is stored in the drive waveform table 102b of the illumination fringe signal generator 102, and the preset value of the counter 102a is stored. The above nine striped illumination states can be realized simply by setting according to the striped illumination state, so that an illumination state for obtaining super-resolution can be generated with a small storage area and a simple configuration. Can be made.

  As described above, one super-resolution image is finally obtained from the nine pieces of image data stored by changing the striped illumination state. A method for obtaining a super-resolution image will be described below.

In a microscope optical system having a point image intensity distribution P (r) at a point r = (x, y) on the image, if the point O (r) on the sample 70 is illuminated with a certain intensity distribution, the sample 70 The laser light (illumination light) in is subjected to spatial modulation. In the case of a sinusoidal illumination having a single spatial frequency component vector K, the spatial modulation components are 0, ± 1st order three modulation components. When these spatial modulations are expressed as m ₁ exp (ilkr + ilφ), (l = 1, 0, −1), the image of the sample 70 subjected to the spatial modulation can be expressed as the following equation (1). Here, r is a position vector on the sample 70, Kr indicates an inner product, and * indicates a convolution integral.

When the image is acquired by changing the fringe illumination as described above, N images (here, 3 images) having the same modulation frequency and modulation amplitude and different only in the modulation phase φ are obtained. When the illumination phase of the j-th image is represented as φ _j , the j-th image is represented by the following equation (2), and N equations are obtained.

  Further, the incident light quantity I (r) is calculated from the input light quantity of the photodetector (second photodetector 53 in the above example) and the previous measurement data of the output signal by the following equation (3) by the polynomial of the output signal S (r). ). In addition, it is desirable to store the previous measurement data in the memory of the control unit 100 as a table.

  The relationship between the incident light quantity and the output signal varies depending on the temperature of the photodetector. Therefore, it is necessary to acquire and approximate data in accordance with the temperature of the photodetector that is actually used. Data can be acquired at each temperature and stored in the memory of the control unit 100 as a table. desirable.

  From the above equations (2) and (3), N equations such as the following equation (4) are obtained.

  In these equations, O (r) exp (ilKr), (l = −1, 0, 1) is an unknown, and can be obtained by acquiring an image of N ≧ 3. When three equations out of N equations (4) are used, the following equation (5) is obtained.

  By solving this equation, O (r) exp (ilKr) * P (r), (l = -1, 0, 1) can be obtained.

  Or you may obtain | require from the image of N> = 4 using the least squares method by a well-known method. Noise can be reduced by using the least square method. In these, l = 0 corresponds to a normal microscopic image that is not super-resolution, and l = ± 1 corresponds to a super-resolution component.

O (r) exp (ilKr) * P (r) and (l = −1, 0, 1) obtained above are O _k (k) P _k (r ₊₁ K) and (l in wave number space, respectively. = -1, 0, 1). Here, O _k (k) is a Fourier transform of the distribution of the illuminant in the specimen 70, and P _k (k) is a transfer function (OTF: Optical Transfer Function) of the microscope optical system when illumination having no intensity distribution is given. ).

  The normal transfer function when illumination with no intensity distribution is given is k = −2 NA / λ to 2 NA / λ with respect to the wavelength λ of light and the numerical aperture NA of the objective lens 36, and thus was obtained above. The sample information O (r) exp (ilKr) * P (r), (l = −1, 0, 1) is k = −2NA / λ−K to 2NA with respect to l = −1, 0, 1. It can be seen that the information includes / λ−K, k = −2NA / λ to 2NA / λ, and k = −2NA / λ + K to 2NA / λ + K. Therefore, since O (r) exp (ilKr) * P (r) as a whole includes information from k = -2NA / λ-K to 2NA / λ + K, a microscopic image with high resolution can be obtained.

  As described above, when a super-resolution image is obtained from nine images obtained by shifting the intensity modulation phase and rotating the illumination state, for example, sectioning performance in the optical axis direction (half-value width) ) Can be 330 nm, and the horizontal resolution (half-value width) can be 100 nm.

  The illumination light (excitation light) that has passed through the specimen 70 is detected by the third photodetector 62 of the third detector 60, and the brightness obtained from the scanning position of the laser light on the specimen 70 and the optical signal is used. A transmission image of the specimen 70 can be obtained. In this case, a transmission image of the specimen 70 can be obtained by superimposing three images (-2π / 3 phase, 0 phase, + 2π / 3 phase images) whose intensity modulation phases of the illumination light are shifted. .

  In the above description, when IR pulse light is used as excitation light (two-photon excitation), the fluorescence emitted from the sample 70 is detected by the first photodetector 43 of the first light detection unit 40, and visible light is detected. When used as excitation light (one-photon excitation), the fluorescence emitted from the specimen 70 is detected by the second photodetector 53 of the second detection unit 50 to obtain a super-resolution image. When light is used as excitation light (one-photon excitation), a spectroscopic device is disposed at the position of the second photodetector 53, and the fluorescence emitted from the sample 70 is detected by spectroscopic analysis for each predetermined wavelength. Each super-resolution image can be obtained. When performing multi-wavelength fluorescence observation, there is a leakage of a plurality of fluorescent components in a certain fluorescent component. Therefore, the above-mentioned nine images are unmixed and a fluorescent image of each single fluorescent sample is obtained. It is preferable to obtain and obtain a super-resolution fluorescent image from the nine single fluorescent images by the above-described processing.

  Note that the intensity of the illumination light described above has been described as being modulated in a sine wave shape. However, when observation is performed by two-photon excitation, as shown in FIG. 9, the square of the intensity distribution becomes a sine wave shape. It is desirable to modulate. Also in this case, drive data divided into a predetermined number (for example, 300) from the intensity distribution shown in FIG. 9 is stored in the drive waveform table 102b, and the reference clock clkA generated by the synchronization signal generator 101 is divided into the number ( 300 data) is counted by the counter 102a, and the data is sequentially read from the drive waveform table 102b and the AOTF 22 is controlled.

  In the above description, the drive waveform for driving the AOTF 22 is converted into discrete values, stored in the drive waveform table 102b, and controlled based on the data of the drive waveform table 102b. The operation of the AOTF driver 104 may be controlled using a signal generator that generates a sine wave and a delay circuit that can delay the phase of the sine wave by an arbitrary phase. The amount of delay is the same as described above.

  In the above description, the light source device 20 has been described with respect to the case where the light source 21 is configured by the light source 21 that emits the laser light and the AOTF 22 that modulates the intensity of the laser light. It is not limited to this. For example, a laser light source that can directly change the intensity of the laser light can be used.

  In addition, the AOTF 22 does not need to have the configuration of the light source device 20, and when IR pulse light is used as excitation light (two-photon excitation), any position can be used as long as it is a space from the light source 21 to the second dichroic mirror 35. Can be arranged.

  The third detector 60 is used to acquire a transmission image by detecting the illumination light (excitation light) that has passed through the specimen 70, but when using IR pulse light as excitation light (two-photon excitation). In order to efficiently acquire fluorescence generated from the specimen 70, it may be used to acquire transmitted fluorescence. In this case, the transmission fluorescence acquisition timing needs to be synchronized with the fluorescence acquisition timing by the first detection unit 40.

DESCRIPTION OF SYMBOLS 10 Scanning microscope 20 Light source device 21 Light source 22 AOTF (intensity modulation element)
DESCRIPTION OF SYMBOLS 30 Scan optical system 31 1st dichroic mirror 32 Scan unit 33 Scan lens 34 2nd objective lens 35 2nd dichroic mirror 36 Objective lens 40 1st detection part 41 1st condensing lens 42 2nd condensing lens 43 1st light detection Device 50 second detector 51 third condenser lens 52 light shielding plate 52a pinhole 53 second photodetector 60 third detector 61 fourth condenser lens 62 third photodetector 70 sample 100 controller 101 synchronization signal generation Unit 102 illumination fringe signal generator 102a counter 102b drive waveform table 103 DA conversion element 104 AOTF driver 105 scanner driver 106 signal amplifier 107 AD conversion element 108 memory I primary image plane P exit pupil

Claims (8)

An illumination optical system that scans a sample using a scanning unit with illumination light emitted from a light source unit;
A condensing optical system for guiding light from the specimen to the detection unit;
A control unit that controls the scanning position of the illumination light in the specimen by the scanning means, and controls the illumination light according to the scanning position so that the intensity change of the illumination light is a 1 / nth power of a sinusoidal waveform; A scanning microscope characterized by comprising:
However, n is an integer of 1 or more. The light source unit includes an acousto-optic element,
The control unit controls the acoustooptic device using a drive signal having a 1 / nth power of a sinusoidal waveform in accordance with the scanning position, whereby the intensity change of the illumination light is increased to a 1 / nth power of the sinusoidal waveform. The scanning microscope according to claim 1, wherein the scanning microscope is controlled to be The light source unit includes a laser light source,
The control unit controls the laser light source according to the scanning position using a 1 / nth power drive signal having a sinusoidal waveform, whereby the intensity change of the illumination light is changed to a 1 / nth power of the sinusoidal waveform. It controls so that it may become. The scanning microscope of Claim 1 characterized by the above-mentioned.   The said control part produces | generates the image of the said sample by calculation from the several image acquired in the illumination state which changed the phase of the said sine wave waveform of the said illumination light, The one of Claims 1-3 characterized by the above-mentioned. The scanning microscope according to one item.   The plurality of images acquired in the illumination state in which the phase of the sinusoidal waveform of the illumination light is changed are in an illumination state in which the phase of the illumination fringe pattern is changed by changing the phase of the sinusoidal waveform of the illumination light. The scanning microscope according to claim 4, wherein the scanning microscope is a plurality of acquired images.   The plurality of images acquired in the illumination state in which the phase of the sinusoidal waveform of the illumination light is changed, the illumination fringe pattern rotated by changing the phase of the sinusoidal waveform of the illumination light for each scanning line The scanning microscope according to claim 4, wherein the scanning microscope is a plurality of images acquired in a state. The controller is
A drive waveform table for storing data for one period of the intensity modulation;
A counter that circulates and reads out the data starting from a position specified as a preset value in the drive waveform table, and sets the preset value according to the illumination state and reads out the sine The scanning microscope according to any one of claims 4 to 6, wherein a wavy waveform is controlled.   8. The apparatus according to claim 1, further comprising a detection unit that is disposed at a position facing the illumination optical system via the sample and detects the illumination light transmitted through the sample. Scanning microscope.

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