JP2011257691A - Laser microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser microscope device capable of changing observation conditions according to a change in sample's status and of performing a high-speed and high-resolution observation.SOLUTION: A laser microscope device 21 to be adopted comprises: a laser irradiation optical system 10 capable of irradiating a sample with CARS excitation light and Raman scattering excitation light in a coaxial manner; a Raman scattering spectrum detector 3 for detecting a spectrum of Raman scattering light generated from a sample 105; a Raman scattering spectrum analyzer 110 for analyzing the spectrum of Raman scattering light and calculating a specific frequency of molecule in the sample; a detuning addition/deletion controller 111 for setting the frequency calculated by the Raman scattering spectrum analyzer 110 as detuning of the CARS excitation light; and a CARS detector 5 for detecting CARS light generated from the sample 105 by irradiating the sample with the CARS excitation light having the set detuning from the laser irradiation optical system 10.

Description

  The present invention relates to a laser microscope apparatus for observing CARS light and Raman scattered light.

  Conventionally, in the study of living cells, observation using a CARS microscope using the principle of coherent anti-Stokes Raman scattering (hereinafter referred to as “CARS”) and observation using a confocal Raman microscope using the principle of confocal effect and Raman scattering. A laser microscope apparatus for performing the above is known (see, for example, Patent Documents 1 and 2).

  A living cell changes with time, and a microscope using the principle of Raman scattering is effective for performing unstained processes from cell division to cell death of living cells. For example, cell division occurs through several states called the cell cycle. In the process leading to changes in the state of cells and cell death, intermediate product molecules appear in living cells. There is a desire to follow such changes in living cells at the molecular level.

  Also, due to changes in the molecular structure of drug molecules and proteins and nucleic acids in living cells when the specimen to be observed, especially live cells, is divided or drug stimulated. It is known that the spectrum of Raman scattered light changes. Therefore, if a Raman scattering microscope is used, it becomes possible to observe the change of a living cell.

  In addition, according to the Raman scattering microscope, the wavelength at which the spectrum of the Raman scattered light is changed by post-processing even when a new peak rises or disappears in the Raman scattered light spectrum of the sample during time-lapse observation. Since an XY image of can be constructed, an observation image following the change of the sample can be obtained.

JP 2009-47435 A JP 2009-58578 A

  However, when observing a specimen with a microscope using the principle of Raman scattering, if the specimen moves or the Raman scattering spectrum of the specimen changes at high speed, images that follow these phenomena are acquired with high resolution and high resolution. It is extremely difficult to do.

  On the other hand, if a CARS microscope is used, high-resolution and high-speed image acquisition is possible. However, in the CARS microscope, an observation image can be obtained only when the frequency difference (hereinafter referred to as “detuning”) of the two-color pulse laser beams is matched with the vibration level of the specimen. Therefore, it is possible to obtain an observation image only with a specimen whose vibration level is clear in advance, even if the Raman scattering spectrum changes or a new vibration level is created, such as during time-lapse observation of live cells, It is impossible to observe the microscope following the change of the specimen.

  The microscope disclosed in Patent Document 1 switches between Raman scattered light observation and CARS light observation without moving the specimen. Therefore, the use of the spectrum of Raman scattered light is limited to the selection of the molecular frequency at which CARS light observation is performed at the start of observation, and CARS light observation that follows changes in the sample cannot be performed.

  The microscope disclosed in Patent Document 2 acquires the Raman scattered light spectrum of a specimen prior to CARS light observation, and sets the excitation laser wavelength and spectral filter conditions in accordance with the molecular frequency of the specimen. is there. Therefore, the use of the spectrum of Raman scattered light is limited at the time of setting the CARS light observation conditions, and CARS light observation following the change of the sample cannot be performed.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a laser microscope apparatus capable of changing observation conditions according to a change in the state of a sample and performing high-speed and high-resolution observation. To do.

In order to achieve the above object, the present invention employs the following means.
The present invention relates to an irradiation optical system capable of irradiating a specimen coaxially with CARS excitation light and Raman scattering excitation light, and a spectrum of Raman scattered light generated from the specimen by irradiating Raman scattering excitation light with the irradiation optical system. A Raman scattered light spectrum detecting means for detecting; a spectrum analyzing means for analyzing a spectrum of the Raman scattered light detected by the Raman scattered light spectrum detecting means to calculate a specific frequency of a molecule in the sample; and the spectrum Detuning setting means for setting the frequency calculated by the analyzing means as detuning of the CARS excitation light, and irradiating the irradiation optical system with the detuned CARS excitation light set by the detuning setting means. A laser microscope apparatus including CARS light detecting means for detecting CARS light generated from the specimen is employed.

  According to the present invention, when the sample is irradiated with Raman scattering excitation light by the irradiation optical system, Raman scattered light is generated from the sample, and its spectrum is detected by the Raman scattered light spectrum detecting means. The detected spectrum of the Raman scattered light is analyzed by the spectrum analyzing means, and a specific frequency of the molecule in the sample is calculated. Then, the detuning setting unit sets the detuning of the CARS excitation light to the frequency calculated by the spectrum analyzing unit. By irradiating the specimen with the CARS excitation light set in this way by the irradiation optical system, CARS light is generated from the specimen and detected by the CARS light detection means.

  By doing as mentioned above, the CARS excitation light detuning can be set according to the spectrum of the Raman scattered light, and the CARS light corresponding to the set detuning can be detected. As a result, CARS light observation that follows the change in the state of the specimen can be performed, and high-speed and high-resolution observation can be performed even when the state of the specimen changes.

In the above invention, a Raman scattered light spectrum storage unit that stores a spectrum of Raman scattered light detected by the Raman scattered light spectrum detection unit is provided, and the detuning setting unit is stored in the Raman scattered light spectrum storage unit. The frequency at which the spectrum of the Raman scattered light changes with time may be set as the detuning of the CARS excitation light.
By doing in this way, the frequency with which the spectrum of the Raman scattered light changed with time can be set as the detuning of the CARS excitation light, and the CARS light can be detected. Thereby, CARS light observation which followed the state change of a sample can be performed.

In the above invention, the detuning setting means may set the frequency at which the intensity of the spectrum of Raman scattered light peaks as the detuning of the CARS excitation light.
By setting the frequency at which the intensity of the spectrum of the Raman scattered light reaches a peak as the detuning of the CARS excitation light, the CARS light can be detected quickly and easily.

In the above invention, the detuning setting means may set the frequency at which the peak of the spectrum of Raman scattered light exceeds a predetermined threshold as the detuning of the CARS excitation light.
When the spectrum intensity of the Raman scattered light is low, CARS light is not detected even if CARS excitation light is detuned again. Therefore, by avoiding the setting of CARS excitation light detuning for the peak of the spectrum of Raman scattered light below a predetermined threshold, unnecessary processing is prevented and stable CARS light observation is performed. It can be carried out.

In the above invention, the Raman scattered light spectrum detecting means disperses the Raman scattered light generated from the sample into a spectrum and a plurality of channels each detecting the spectrum of the Raman scattered light dispersed by the spectroscopic section It is also possible to have a multi-channel detector.
By doing in this way, the Raman scattered light generated from the sample can be split into spectra by the spectroscopic unit, and these spectra can be detected by the multi-channel detector, respectively.

In the above invention, the irradiation optical system may irradiate Raman scattering excitation light with a smaller number of irradiation points than CARS excitation light.
By doing in this way, the number of data points of the Raman scattered light detected by the Raman scattered light spectrum detection means can be made smaller than the number of data points of the CARS light detected by the CARS light detection means. Here, the Raman scattered light only needs to be able to set the detuning of the CARS excitation light, so the number of data points may be small. Therefore, by performing as described above, the processing at the time of setting the detuning of the CARS excitation light can be facilitated, and CARS light observation following the state change of the sample can be performed at high speed.

In the above invention, a lens for modulating the wavefront of the excitation light source may be provided on the optical path of the Raman scattering excitation light source, and the sample may be surface illuminated by the irradiation optical system.
By doing so, the surface of the sample can be irradiated with Raman scattering excitation light, and the Raman scattering spectrum of the sample can be acquired in a lump, thereby enabling higher-speed measurement.

In the above invention, the Raman scattered light spectrum detecting means is a fiber spectroscope including a light receiving unit using an optical fiber, and a spectroscopic unit that splits light received by the optical fiber, wherein the light receiving unit includes: It may be arranged at a position conjugate with the pupil of the objective lens.
By using the Raman scattered light spectrum detection means as a fiber spectrometer and arranging the light receiving portion of the optical fiber at a position conjugate with the pupil of the objective lens, the Raman scattered light collected by the objective lens can be taken in at once.

In the above invention, a time lapse observation means for performing time lapse observation may be provided.
By doing in this way, a time lapse observation image can be obtained by the time lapse observation means, and the change of the sample can be observed.

In the above-described invention, image acquisition means for acquiring a preview image for designating an observation position may be provided.
In this way, it is possible to easily set the observation area from the preview image.

  According to the present invention, it is possible to perform observation at high speed and with high resolution by changing the observation conditions in accordance with the state change of the specimen.

1 is an overall configuration diagram of a laser microscope apparatus according to a first embodiment of the present invention. It is a block diagram which shows the structure of the CARS excitation light source of FIG. It is a block diagram which shows the structure of the Raman scattering excitation light source of FIG. It is a graph which shows the wavelength characteristic of the dichroic mirror of FIG. It is a block diagram which shows the structure of the CARS detection part of FIG. It is a graph which shows the optical characteristic of the band pass filter of FIG. It is a block diagram which shows the structure of the Raman scattering spectrum detection part of FIG. It is a flowchart which shows the process in the observation condition setting phase by the laser microscope apparatus of FIG. It is an example of the preview image acquired by the laser microscope apparatus of FIG. 1, (a) is before a Raman scattering spectrum acquisition range setting, (b) is after a Raman scattering spectrum acquisition range setting. It is a flowchart which shows the process in the observation phase by the laser microscope apparatus of FIG. It is an example of the graph which shows the time change of a Raman scattering spectrum. It is a list (an example) of the image acquired by CARS observation. It is a whole block diagram of the laser microscope apparatus which concerns on the 2nd Embodiment of this invention. It is a block diagram which shows the structure of the Raman scattering excitation light source of FIG. It is a block diagram which shows the structure of the Raman scattering spectrum detection part of FIG. It is an example of the preview image acquired by the laser microscope apparatus of FIG.

[First Embodiment]
The laser microscope apparatus 21 according to the first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a laser microscope apparatus 21 according to the present embodiment includes a CARS excitation light source 1 that emits CARS excitation light, a Raman scattering excitation light source 2 that emits Raman scattering excitation light, and samples light from these light sources. A laser irradiation optical system (irradiation optical system) 10 that irradiates 105, a Raman scattering spectrum detection unit (Raman scattering light spectrum detection means) 3 that detects Raman scattering light, and a CARS detection unit (CARS light detection) that detects CARS light Means) 5 and a control unit 6 for controlling them.

The CARS excitation light source 1 is a light source that emits CARS excitation light for generating CARS light from the specimen 105 to be observed. As shown in FIG. 2, the first pulse laser light source 1a, the second pulse laser light source 1b, The reflection mirror 1c and the dichroic mirror 1d are provided.
The characteristic of the dichroic mirror 1d is configured to transmit only the wavelength of the first pulse laser light source 1a and reflect the wavelength of the second pulse laser light source 1b.

  In the CARS excitation light source 1, the laser light output from the first pulse laser light source 1 a is emitted from the CARS excitation light source 1 through the dichroic reflection mirror 1 d. The laser light output from the second pulse laser light source 1b is reflected by the reflection mirror 1c, then reflected by the surface of the dichroic mirror 1d, and is synthesized and emitted coaxially with the laser light from the first pulse laser light source 1a. The By having the above configuration, the CARS excitation light source 1 emits laser light having two colors of wavelengths from the first pulse laser light source 1a and the second pulse laser light source 1b coaxially and emits them as CARS excitation light. It has become.

In this embodiment, the first pulse laser light source 1a is a fixed wavelength pulse laser, and the second pulse laser light source 1b is a variable wavelength pulse laser, so that the two-color pulse laser emitted from the CARS excitation light source 1 is used. The light frequency difference (hereinafter referred to as “detuning”) Δω can be adjusted according to the frequency ω _q of elementary excitation in the specimen 105 to be observed. Here, as the second pulse laser light source 1b, a variable wavelength pulse laser light source such as a titanium sapphire laser is used.

  The Raman scattering excitation light source 2 is a light source that emits Raman scattering excitation light for generating Raman scattered light from the specimen 105. As shown in FIG. 3, near the monochromatic continuous wave laser light (for example, wavelength 748 nm or 785 nm). It has a Raman scattering excitation light source 2a which is an infrared LD.

  As shown in FIG. 1, the laser irradiation optical system 10 includes a reflection mirror 117, a dichroic mirror 118, an optical scanning unit 4, a pupil projection lens 100, a reflection mirror 122, an imaging lens 101, and an objective lens 102. And. The laser irradiation optical system 10 irradiates the specimen 105 with CARS excitation light emitted from the CARS excitation light source 1 and Raman scattering excitation light emitted from the Raman scattering excitation light source 2.

  The optical scanning unit 4 is a galvanometer mirror, for example, and includes a deflection mirror 4a and a deflection mirror 4b. The rotation axes of the deflecting mirrors are configured to be orthogonal to each other. Further, the rotation axis of each deflection mirror and the optical axis of the incident laser light are configured to be orthogonal to each other. Further, when the swing angle of each deflection mirror is 0 degree, the angle formed by the reflection surface of each deflection mirror and the optical axis direction of the laser beam is 45 degrees.

  The wavelength characteristics of the dichroic mirror 118 are such that the CARS excitation light from the CARS excitation light source 1 is transmitted while the Raman scattering excitation light from the Raman scattering excitation light source 2 is reflected. Further, as shown in FIG. 4, the wavelength characteristic of the dichroic mirror 121 transmits the CARS excitation light from the CARS excitation light source 1 and the Raman scattering excitation light from the Raman scattering excitation light source 2, while the Stokes Raman from the sample 105 is transmitted. The characteristic is to reflect scattered light.

  With the above configuration, the CARS excitation light emitted from the CARS excitation light source 1 passes through the dichroic mirror 118 and the dichroic mirror 121 and is guided to the optical scanning unit 4. The CARS excitation light guided to the optical scanning unit 4 is reflected by the deflection mirrors 4 a and 4 b and then passes through the pupil projection lens 100. The CARS excitation light transmitted through the pupil projection lens 100 is folded 90 degrees by the reflection mirror 122, transmitted through the imaging lens 101 and the objective lens 102, and placed on the focusing stage 103 that can move up and down in the optical axis direction. The sample 105 is collected on the sample surface 104.

  Further, the Raman scattered excitation light emitted from the Raman scattering excitation light source 2 is reflected by the reflection mirror 117, then reflected by the surface of the dichroic mirror 118, and folded back in the optical axis direction of the CARS excitation light. The Raman scattered excitation light is synthesized coaxially with the CARS excitation light, passes through the dichroic mirror 121, passes through the optical scanning unit 4, and passes through the pupil projection lens 100. The Raman scattered excitation light that has passed through the pupil projection lens 100 is folded 90 degrees by the reflection mirror 122, passes through the imaging lens 101 and the objective lens 102, and is collected on the specimen surface 104 of the specimen 105 placed on the stage 103. To be lighted.

In the present embodiment, since the CARS light is detected by the transmission type, the CARS detector 5 is disposed on the incident light transmission side with respect to the specimen 105.
The CARS light generated in the specimen 105 passes through the lens 123 disposed on the incident light transmission side with respect to the specimen 105, is turned back by 90 degrees by the reflection mirror 124, and can be inserted into and removed from the optical path (for example, rotated). Is transmitted through the band-pass filter 106 held by the filter turret) and guided to the CARS detector 5.

  As shown in FIG. 6, the bandpass filter 106 has an optical characteristic of reflecting only the CARS light while reflecting the wavelength of the CARS excitation light. The CARS light that has entered the CARS detection unit 5 passes through the condenser lens 125 and enters the photodetector 107.

  As shown in FIG. 5, the CARS detector 5 includes a condenser lens 125 that condenses the CARS light generated in the specimen 105 on the photodetector 107, and light that detects the CARS light collected by the photodetector 107. A detector (for example, a photomultiplier tube) 107 is provided.

On the other hand, in this embodiment, the detection of the Raman scattered light is performed by a reflection type, and therefore the Raman scattered spectrum detection unit 3 is arranged on the incident light reflection side with respect to the specimen 105.
The Raman scattered light generated from the sample 105 traces the path of the Raman scattering excitation laser in reverse. That is, the Raman scattered light generated from the specimen 105 is transmitted through the objective lens 102 and the imaging lens 101, turned back 90 degrees by the reflection mirror 122, passed through the pupil projection lens 100 and the optical scanning unit 4, and then to the dichroic mirror 121. Finally, it is turned 90 degrees and guided to the Raman scattering spectrum detector 3.

  As shown in FIG. 7, the Raman scattering spectrum detection unit 3 reflects the CARS excitation light and the Raman scattering excitation light, while transmitting only the Raman scattered light and the band pass filter 3a. And a condensing lens 3b for condensing the Raman scattered light that has been collected onto the slit portion 3c of the spectroscopic detector 3A, and a spectroscopic detector 3A for detecting the Raman scattered light spectrum.

  The spectroscopic detector 3A connects a concave mirror 3d that receives light that has passed through the slit 3c, a grating (spectral part) 3e that spectrally decomposes incident light, and a spectral component that is spectrally separated by the grating 3e to the subsequent line detector 3g. It has a concave mirror 3f for imaging, and a line detector (multi-channel detector) 3g for detecting spectral components.

  The line detector 3g has, for example, a multi-channel photomultiplier tube in which a plurality of photomultiplier tubes are arranged in a line shape. In this embodiment, the line detector 3g adopts a spectroscopic detection method called a Zelni Turner arrangement.

  As shown in FIG. 1, the control unit 6 includes an image generation unit 126 that generates an image of the specimen 105, a detuning addition / deletion control unit (detuning setting unit) 111 that sets detuning of CARS excitation light, A Raman scattering spectrum analysis unit (spectrum analysis means) 110 for analyzing the Raman scattering spectrum.

The image generation unit 126 generates an image of the sample 105 from the CARS light from the sample 105 detected by the CARS detection unit 5.
The Raman scattering spectrum analyzing unit 110 analyzes the spectrum of the Raman scattered light detected by the Raman scattered light spectrum detecting unit 3 and calculates a specific frequency of molecules in the sample 105.
The detuning addition / deletion control unit 111 sets the frequency calculated by the Raman scattering spectrum analysis unit 110 as the detuning of the CARS excitation light.

  A CARS excitation light source 1, a Raman scattering excitation light source 2, an optical scanning unit 4, a CARS detection unit 5, and a Raman scattering spectrum detection unit 3 are connected to the control unit 6 via a communication interface. Further, for example, an input device 112 such as a mouse or a keyboard and an external output device 113 such as a monitor are connected to the control unit 6.

The control unit 6 has a Raman scattering excitation light source control function, a CARS excitation light source control function, a Raman scattering acquisition point storage function, a CARS microscope observation image acquisition area storage function, and a CARS excitation light source detuning setting storage function (not shown). is doing.
The Raman scattering spectrum analysis unit 110 includes a storage function that associates and holds a plurality of Raman scattering spectra, information on a position where the Raman scattering spectrum is detected, and a detected time, a function that detects a spectrum peak, and a plurality of Raman scattering spectra. And a function of detecting changes in peak frequency and intensity in comparison with each other.

A microscope observation method using the laser microscope apparatus 1 having the above-described configuration will be described separately for an observation condition setting phase shown in FIG. 8 and an observation phase shown in FIG.
Hereinafter, the procedure for setting the observation condition will be described with reference to the flowchart of the observation condition setting phase shown in FIG.

First, in step S1 (acquisition of a preview image), a preview image for setting a specimen observation condition is created. The acquisition of the preview image is performed by observation with a Raman microscope using the Raman scattering excitation light source 2 and the laser irradiation optical system 10 with the optical scanning illumination of the sample 105 and the light microscope 107.
At this time, the band-pass filter 106 is removed from the optical path so that the laser light transmitted through the sample 105 enters the photodetector 107.

Next, details of transmission microscope observation using the above-described apparatus will be described.
The specimen 105 is illuminated by laser light emitted from the Raman scattering excitation light source 2 according to a command from the control unit 6 to the imaging lens 101 and the objective lens 102 via the optical scanning unit 4, pupil projection lens 100, and reflection mirror 122. Then, the specimen 105 is two-dimensionally scanned by the rotating deflection mirror pairs 4a and 4b of the optical scanning unit 4.

  The illumination light that has passed through the specimen 105 passes through the lens 123, changes its direction by the reflection mirror 124, is guided to the photodetector 107, and the photodetector 107 detects the intensity of the light. The control unit 6 associates the angle information of the deflection mirrors 4 a and 4 b corresponding to each illumination point on the specimen 105 and the luminance information detected by the photodetector 107 and outputs the associated information to the image generation unit 126.

  The image generation unit 126 generates an image based on the position information and the luminance information, and outputs the image information to the external output device 113. The external output device 113 displays the transmission microscope observation image as a preview image on the screen.

  Here, FIG. 9A and FIG. 9B show an example of the preview image acquired by the above method and an example of settings performed in step S2 and step S3 described later. In FIG. 9A and FIG. 9B, the range surrounded by the chain line shows the range observed by the CARS microscope with 512 × 512 pixels as an example. On the other hand, a point indicated by a black point indicates a point at which a Raman scattering spectrum is acquired. As shown in this example, the number of points from which the Raman scattering spectrum is acquired is set to be smaller than the number of pixels observed with the CARS microscope. Note that the acquired Raman scattering spectrum is not the image itself, and thus does not need to be taken at many points as in the image acquisition. Therefore, the acquisition time of the Raman scattering spectrum can be shortened.

In step S <b> 2 (CARS image acquisition region setting), an image acquisition region by CARS microscope observation is set on the preview image using the input device 112.
In step S3 (Raman scattering spectrum measurement point setting), a Raman scattering spectrum measurement point is designated on the preview image. The measurement point designation method uses the input device 112 to input a plurality of characteristic points in the cell so that a Raman scattering spectrum can be obtained without leakage even when molecules are localized in the sample. Set. Alternatively, as shown in FIG. 9B, for the purpose of setting the measurement points of the Raman scattering spectrum quickly and uniformly, the measurer only specifies the number of measurement points and the measurement range (draws the ROI). Specific measurement points may be set randomly by the control unit 6.

In step S4 (time lapse observation time setting), time lapse observation time (interval and number of times) performed in the observation phase is set.
As described above, the observation condition setting phase is completed by the operation from step S1 to step S4, and the process proceeds to the observation phase shown in the flowchart of FIG.

Below, the procedure in an observation phase is demonstrated using FIG.
In step S5 (Raman scattering spectrum measurement), the optical scanning unit 4 adjusts the angles of the deflection mirrors 4a and 4b so as to correspond to the Raman scattering spectrum acquisition point set in step S3 according to a command from the control unit 6. On the other hand, the Raman scattering excitation light source 2 emits Raman scattering excitation light. The Raman scattered light generated from the specimen 105 by being irradiated with the Raman scattered excitation light is guided to the Raman scattered spectrum detection unit 3.

  In the Raman scattering spectrum detector 3, after the Raman scattering excitation light is cut by the bandpass filter 3a, an image is formed at the position of the slit 3c of the spectroscopic detector 3A by the lens 3b. The Raman scattered light that is diffracted and spread from the slit 3c is reflected by the concave mirror 3d to be converted into parallel light and dispersed by the grating 3e, and then reflected by the concave mirror 3f to form an image on the line detector 3g. When each light receiving element constituting the line detector 3g detects Raman scattered light, it outputs it to the control unit 6 as a Raman scattered spectrum signal. The control unit 6 makes one Raman scattering spectrum signal obtained by integrating the Raman scattering spectrum signals acquired at each point as described above.

In step S6 (Raman scattering spectrum storage), the Raman scattering spectrum signal integrated in the Raman scattering spectrum analysis unit 110 and the reception time are stored in association with each other.
In step S7 (CARS detuning setting from Raman scattering spectrum), the frequency to be excited by CARS is selected from the Raman scattering spectrum acquired in step S5. At this time, it may be set manually or may be automatically set by detecting the peak frequency with the Raman scattering spectrum analysis unit 110. In the case of manual operation, the frequency to be excited by CARS is specified using the input device 112. The control unit 6 calculates the frequency difference between the two colors of laser light emitted from the CARS excitation light source so as to match the designated detuning, and determines the frequency (wavelength) of the laser light source 1b (wavelength variable laser). adjust.

  In step S <b> 8 (image acquisition by CARS microscope observation), the control unit 6 causes CARS excitation light to be emitted from the CARS excitation light source 1. On the other hand, the optical scanning unit 4 scans the CARS microscope observation region set in step S2 by swinging the deflection angles of the deflection mirrors 4a and 4b. CARS light is generated from the sample 105 thus excited, and the CARS light is incident on the CARS detector 5 through the lens 123, the reflection mirror 124, and the bandpass filter 106. Thereafter, the CARS light passes through the condenser lens 125 and is detected by a photomultiplier tube (not shown) of the photodetector 107. The control unit 6 associates the position information with the luminance signal detected by the spectroscopic detector 107 and outputs it to the image generation unit 126. The image generation unit 126 generates an image from the position information and the luminance signal, and then outputs the image to the control unit 6 to store the image.

In step S9 (time lapse time check), the control unit 6 compares the elapsed time of the time lapse observation with the time lapse observation time set in step S4, and determines whether or not the time lapse measurement is continued.
In step S17 (end), if the set time has elapsed in step S9, the time lapse observation is ended.

In step S10 (Raman scattering spectrum measurement), if it is determined in step S9 that the time lapse measurement is continued, the same processing as in step S5 is performed.
In step S11 (Raman scattering spectrum storage), the same processing as in step S6 is executed.

In step S12 (Raman scattering spectrum mutual comparison analysis), the Raman scattering spectrum analysis unit 110 compares the stored Raman scattering spectrum information with each other, and performs a mutual comparison of the peak frequency and the peak intensity.
In step S13 (whether the peak frequency for which detuning is set falls below a threshold value), it is determined in the analysis result in step S12 whether the peak intensity of the frequency for which CARS detuning is set is below the threshold value. To do. If it is not below, nothing is done and the process proceeds to step S15.

In step S14 (detuning is deleted), in the determination in step S13 above, when the frequency falls below the threshold, the CARS detuning setting for the frequency is deleted.
In step S15 (whether a new peak has appeared in the Raman scattering spectrum), it is determined whether a new peak has appeared in the Raman scattering spectrum in the analysis result in step S12. If NO is determined in step S15, nothing is done and the process returns to step S8.

In step S16 (additional setting of detuning), if YES is determined in the determination of step S15, CARS detuning is additionally set to the newly appearing peak frequency, and the process returns to step S8.
In step S18 (stimulation is performed), in the above-described observation example, when stimulation such as drug administration is performed on the specimen, it is performed after step S8.

  In the above description, in step S16, CARS detuning is added to the newly appearing peak frequency. However, CARS detuning is added to the frequency at which a decrease in Raman spectrum intensity is observed. If the CARS detuning is matched with the “Raman changed frequency”, a remarkable phenomenon of the sample 105 can be captured as a CARS image.

The observation result of the specimen 105 whose Raman scattering spectrum changes with time in the configuration of the present embodiment will be described with reference to FIGS.
FIG. 11 shows the Raman scattering spectra of the sample 105 acquired from time T1 to T4 and displayed side by side. As an addition / deletion operation of CARS detuning by analysis of Raman scattering spectrum, the peak frequency newly generated at time T3 is added to the detuning of CARS microscope observation, and the peak falls below the threshold at time T3 and time T4. Remove frequency detuning.

  FIG. 12 shows the result of CARS microscope observation from time T1 to T4 described above. At the beginning of observation, CARS was detuned to peak frequency A and peak frequency B, and images were acquired. After that, detuning was additionally set at the peak that appeared at time T3 to acquire an image, and detuning A was deleted at time T3 to stop image acquisition. Further, the setting of detuning B is deleted at time T4.

  In this way, in this embodiment, addition or deletion is performed while taking CARS, but at first, when Raman scattering is taken with time lapse and an intensity change is detected, it is used as a trigger. The CARS detuning may be set to the changed frequency, and the CARS microscope observation may be started for the first time. By doing in this way, the change of the sample 105 can be captured as a CARS image, and an effect of reducing damage to the sample 105 can be obtained.

The effect of the laser microscope apparatus 1 in which each process is performed as described above will be described.
According to the laser microscope apparatus 21 according to the present embodiment, CARS microscope observation that enables high-speed image acquisition is performed by exciting the vibration level of the specimen 105 with two-color pulse laser light (CARS excitation light). Can do.

  Prior to CARS microscope observation, a Raman scattering spectrum is obtained with a smaller number of data points than the number of pixels at the time of CARS microscope observation, and the change in the spectrum is analyzed. Even if it changes, the CARS detuning can be quickly added to the changed frequency. As a result, it is possible to acquire a high-resolution image that follows the change at high speed.

  Therefore, it is possible to carry out a microscope observation in which the CARS microscope observation, which is difficult to follow the change in the Raman scattering spectrum of the sample, and the problem of the Raman scattering microscope, which takes a long time to acquire a microscope observation image, are compensated for each other.

In addition, since the Raman scattering spectrum is acquired at a plurality of points, the Raman scattering spectrum can be acquired without omission even when molecules are localized in the sample 105.
In addition, since the measurement points can be set automatically only by specifying the number of measurement points of the Raman scattering spectrum and the measurement range, the measurement points can be set quickly and uniformly.

In addition to adding the detuning of CARS microscope observation, the detuning is also deleted following the disappearance of the peak, so unnecessary illumination is eliminated and the invasion to the specimen 105 is minimized. Can be suppressed.
Since the preview image of the specimen 105 is acquired prior to the start of observation, the Raman scattering spectrum acquisition point and the CARS microscope observation area in the specimen 105 can be easily set.

In this embodiment, the detuning addition / deletion control unit 111 sets the frequency at which the spectrum of the Raman scattered light has changed with time as the detuning of the CARS excitation light, but instead of this, the spectrum of the Raman scattered light is changed. The frequency at which the intensity reaches a peak may be set as the detuning of the CARS excitation light.
Thus, by setting the detuning of the CARS excitation light, the CARS light can be detected easily at high speed.

Further, the detuning addition / deletion control unit 111 may set the frequency at which the intensity of the spectrum of the Raman scattered light reaches a peak exceeding a predetermined threshold as the detuning of the CARS excitation light.
When the spectrum intensity of the Raman scattered light is low, CARS light is not detected even if CARS excitation light is detuned again. Therefore, by avoiding the setting of CARS excitation light detuning for the peak of the spectrum of Raman scattered light below a predetermined threshold, unnecessary processing is prevented and stable CARS light observation is performed. It can be carried out.

[Second Embodiment]
The laser microscope apparatus 22 according to the second embodiment will be described below with reference to FIG. Hereinafter, regarding the laser microscope apparatus 22 according to the present embodiment, the points common to the laser microscope apparatus 21 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different points are mainly described.

  As shown in FIG. 13, the laser microscope apparatus 22 according to the present embodiment includes a CARS excitation light source 1 that emits CARS excitation light, a Raman scattering excitation light source 7 that emits Raman scattering excitation light, and samples light from these light sources. A laser irradiation optical system (irradiation optical system) 10 that irradiates 105, a Raman scattering spectrum detection unit (Raman scattering light spectrum detection means) 8 that detects Raman scattering light, and a CARS detection unit (CARS light detection) that detects CARS light Means) 5 and a control unit 6 for controlling them.

  As shown in FIG. 14, the Raman scattering excitation light source 7 has a monochromatic continuous wave laser beam, for example, a Raman scattering excitation light source 7a which is a near infrared LD having a wavelength of 748 nm or 785 nm, and at least on the optical path of the monochromatic laser beam. And a wavefront modulation unit 7b capable of adjusting the distance between the two lenses.

The optical axis of the CARS excitation light source 1 and the optical axis of the Raman scattering excitation light source 7 are synthesized coaxially by a dichroic mirror 118.
The Raman scattering excitation light from the Raman scattering excitation light source 7 passes through the pupil scanning lens 100 through the optical scanning unit 4, changes the optical path by 90 degrees with the dichroic mirror 121, and then passes through the imaging lens 101. The Raman scattered excitation light transmitted through the imaging lens 101 is surface-illuminated on the specimen surface 104 of the specimen 105 placed on the stage 103 through the objective lens 102.

  On the other hand, the CARS excitation light from the CARS excitation light source 1 passes through the dichroic mirror 118, passes through the optical scanning unit 4, passes through the pupil projection lens 100, and then is reflected by the dichroic mirror 121 to change the direction of the 90-degree optical path. Instead, the light passes through the imaging lens 101 and the objective lens 102 and is condensed on the specimen surface 104.

On the separation optical path of the dichroic mirror 121, the Raman scattering spectrum detector 8 is arranged.
As shown in FIG. 15, the Raman scattering spectrum detection unit 8 includes a bundle fiber 8a, a condensing lens 8b, and a spectral detector 3A.
Between the dichroic mirror 121 and the Raman scattering spectrum detection unit 8, there is a pupil projection lens 127 for projecting the pupil of the objective lens 102 onto the incident end face 8 c of the bundle fiber, and a characteristic of transmitting only the wavelength of the Raman scattered light. A bandpass filter 128 is arranged.

The position of the incident end face 8 c of the bundle fiber 8 a is arranged so as to be a conjugate position with the pupil of the objective lens 102.
On the other hand, a spectroscopic detector 3A is disposed on the exit end 8e side of the bundle fiber 8a via a condenser lens 8b.

  The bundle fiber 8a has a shape in which the incident end 8c is formed in a circular shape as indicated by reference numeral 8d, and the fiber bundle is linearly arranged so that the output end 8e is indicated in reference numeral 8f. Thereby, the light emitted from the emission end 8e is condensed on the entrance slit 3c of the spectroscopic detector 3A by the condenser lens 8b.

  The entrance slit 3c is arranged in a direction orthogonal to the dispersion direction of the grating 3e. Further, the arrangement direction of the fiber bundle at the exit end 8e of the bundle fiber 8a is parallel to the entrance slit 3c.

The operation of the laser microscope apparatus 22 having the above configuration will be described below. Here, portions different from the operations described in the first embodiment will be mainly described, and the same operations will be omitted.
In the laser microscope apparatus 22 according to the present embodiment, when the inter-lens distance of the wavefront modulation unit 7b disposed in the optical path of the Raman scattering excitation light source 7 is changed in the optical axis direction, the wavefront curvature of the Raman scattering excitation light is continuous. To change. Thereby, the laser spot diameter on the specimen 105, that is, the illumination range of the Raman scattered excitation light on the specimen 105 changes.

  In this case, when acquiring the Raman scattering spectrum, the specimen 105 is illuminated with a surface, and when obtaining a preview image by observation with a transmission microscope, the wavefront of the laser light is adjusted so that the specimen 105 is illuminated with a point.

  The Raman scattered light generated by surface illumination of the specimen 105 by the Raman scattering excitation light source 7 traces the optical path of the Raman scattering excitation light in the reverse direction. That is, the Raman scattered light passes through the objective lens 102, the imaging lens 101, and the dichroic mirror 121 and passes through the pupil projection lens 127 and the bandpass filter 128 arranged on the extension of the optical axis. Is guided to.

  The Raman scattered light guided in this way is captured by the incident end face 8c of the bundle fiber 8a, passes through the fiber, is emitted from the exit end face 8e of the fiber, and is detected by the condenser lens 8b. The image is formed on the entrance slit 3c. In the same way as in the first embodiment, the light incident from the entrance slit 3c is converted into parallel light by the concave mirror 3d, dispersed by the grating 3e, and then imaged and detected by the concave mirror 3f on the line detector 3g. Is done.

  In the observation condition setting phase in the present embodiment, the wavefront modulation unit 7b is adjusted so that the illumination range of the Raman scattered excitation light becomes the entire field of view of the preview image as shown by the two-dot chain line portion in FIG. A range surrounded by a chain line indicates a range observed by a CARS microscope with 512 × 512 pixels as an example. In this way, the Raman scattering spectrum is acquired collectively by specifying the region.

The effect of the laser microscope apparatus 22 that exhibits the above-described operation will be described below.
According to the laser microscope apparatus 22 according to the present embodiment, since the wavefront modulation unit 7b is arranged on the optical path of the Raman scattering excitation light source 7a, the surface of the sample 105 can be illuminated with a surface. Therefore, even when molecules are localized in the specimen 105, a Raman scattering spectrum can be acquired without leakage. Furthermore, since a Raman scattering spectrum can be obtained with a single exposure, rapid measurement is possible.

  In addition, since the incident end face 8c of the bundle fiber 8a is arranged at a position conjugate with the pupil of the objective lens 102, even unscanned light can be taken into the bundle fiber 8a without leakage. Further, the Raman scattered light is incident in a two-dimensionally deflected state at the incident end face 8c of the bundle fiber, but the fiber bundle is formed linearly at the outgoing end face 8e, so that the incident slit 3c of the spectral detector 3A is formed. The light collected by the objective lens 102 can be guided to the spectral detector 3A without waste, and bright spectral detection can be performed.

[First Modification]
The laser microscope apparatus according to the first modification will be described below.
In the first embodiment, the time lapse observation is performed without changing the acquisition point position of the Raman scattering spectrum. However, the acquisition point position may be changed every time the observation interval is finished.

A specific method for changing the acquisition point position will be described below.
The control part 6 should just perform the operation | movement which resets the said acquisition point position to a different random position within the range which drawn ROI for every interval.
The timing for changing the measurement point position is immediately before step S10 in FIG. Therefore, as a matter of course, in this modification, in step S10, the Raman scattering spectrum is acquired at the newly set position.

  Here, as the excitation laser light having a normal Raman scattering spectrum, one having a strong power is used in order to quickly acquire the spectrum. On the other hand, according to the laser microscope apparatus according to the present modification, the invasion to the specimen 105 can be minimized by changing the measurement point for each interval.

[Second Modification]
The laser microscope apparatus according to the second modification will be described below.
When the specimen 105 to be observed moves during the measurement, the measurer only specifies the cell of interest and the number of Raman scattering spectrum measurement points, and performs the CARS microscope observation range performed in step S2 of FIG. The setting of the Raman scattering spectrum measurement point position may be dynamically changed by the control unit 6 according to the position and shape of the cell.

At this time, the recognition of the outer shape and position of the cell may be performed by cell tracking software (see, for example, JP-A-2006-209698). If the displacement or deformation of the specimen is not detected as a result of analysis by software, the measurement is performed in the set area and point position.
Further, even if the displacement or deformation of the specimen is not detected, the acquisition point position of the Raman scattering spectrum may be changed randomly every time the observation interval is finished.

  The analysis of the cell position and shape change, the resetting of the CARS microscope observation range, and the resetting operation of the Raman scattering spectrum measurement point will be described below. The analysis of the sample is performed using the preview image, and the same image is acquired for each interval immediately before the time-lapse observation procedure step S10.

  Next, when the acquired images are analyzed to detect the displacement and deformation of the specimen, the ROI is drawn by the analysis, and the CARS microscope observation range is reset and the Raman scattering spectrum measurement point position is reset within the range. Set up. At this time, the Raman scattering spectrum measurement point position is reset at random as the number of acquired points.

  Therefore, naturally, the CARS microscope observation image acquisition in step S8 and the Raman scattering spectrum measurement in step S10 are performed at the newly reset region and point position, respectively.

According to the laser microscope apparatus according to this modification, since the movement and shape change of the cell can be recognized by the control unit 6, the CARS microscope observation range and the Raman scattering spectrum acquisition point can be used for the displacement and deformation of the specimen. It can always be set optimally.
Furthermore, if the acquisition point position of the Raman scattering spectrum is changed to a different random position at each time lapse observation interval, the same effect as that of the first modification example described above can be obtained.

In each of the above-described embodiments and modifications, the transmission microscope observation image is used to acquire the preview image. However, a phase contrast microscope observation image, a differential interference image, or a fluorescence image may be used.
When a phase contrast microscope observation image is used as a preview image, the objective lens is provided with a phase film, and a phase ring that can be inserted and removed is disposed in the condenser lens 123 using, for example, a phase difference capacitor. When carrying out CARS microscope observation after completing the acquisition of the preview image, the microscope can be observed without losing the amount of light if the phase ring is removed.

Further, when the differential interference image is used as a preview image, a Nomarski prism that can be inserted and removed may be disposed on the light source side of the objective lens.
When using the fluorescence image as a preview image, the laser light for fluorescence excitation is synthesized coaxially with the Raman scattering excitation laser, and the Raman scattering spectrum detection unit 3 is provided with a confocal lens and a confocal pinhole. A filter wheel may be provided at the position of the filter 3b so that the filter having the characteristic of transmitting only the fluorescence wavelength to be detected and the bandpass filter 3b can be interchanged. In such a case, since a high-resolution fluorescent image can be used as a preview image, the setting of the Raman scattering spectrum acquisition point and the setting of the acquisition region of the CARS microscope observation image can be performed in more detail. it can.

[Third Modification]
The laser microscope apparatus according to the third modification will be described below.
In the first embodiment, the acquisition of the Raman scattering spectrum and the CARS microscope observation are alternately performed, but may be performed simultaneously. As will be described later, even when optical scanning is performed by the optical scanning unit 4, the optical axis of the Raman scattered light incident on the Raman scattering spectrum detection unit 3 is not shaken, so that the Raman scattered light observation and the CARS microscope observation may be performed simultaneously. Is possible. In this case, laser light is simultaneously emitted from the CARS excitation light source 1 and the Raman scattering excitation light source 2 and condensed at the same point on the specimen 102 placed on the stage 103.

  At this time, even if the excitation laser beam is scanned by the optical scanning unit 4, the Raman scattered light traces the optical path of the excitation laser beam in reverse (descanning), so that the Raman scattered light incident on the Raman scattering spectrum detection unit 3 The optical axis does not shake. Therefore, the spectral characteristics are not deteriorated due to the deviation of the optical axis.

  By shortening the Raman scattering excitation time per point to be scanned, the obtained spectrum becomes weak, but it is sufficient to increase the output of the excitation light accordingly. The spectrum signals obtained at each point are integrated as described above to form one Raman scattering spectrum. Thereafter, the Raman scattering spectra acquired at each time of time lapse observation are comparatively analyzed to detect a change. It is performed in the same manner as in the first embodiment except that the CARS microscope observation and the Raman scattering spectrum acquisition timing are simultaneous.

  According to the laser microscope apparatus according to this modification, the CARS microscope observation and the Raman scattering spectrum acquisition are performed at the same time. Therefore, the microscope observation can be performed faster than the case of performing each separately.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to the above-described embodiments and modifications, and may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is particularly limited. is not.

DESCRIPTION OF SYMBOLS 1 CARS excitation light source 2 Raman scattering excitation light source 3 Raman scattering spectrum detection part (Raman scattered light spectrum detection means)
4 optical scanning part 5 CARS detection part (CARS light detection means)
6 Control unit 7 Raman scattering excitation light source 8 Raman scattering spectrum detection unit (Raman scattered light spectrum detection means)
10 Laser irradiation optical system (irradiation optical system)
21 Laser microscope device 22 Laser microscope device 105 Sample 110 Raman scattering spectrum analysis unit (spectrum analysis means)
111 Detuning addition / deletion control unit (Detuning setting means)
126 Image generation unit

Claims (10)

An irradiation optical system capable of irradiating a specimen coaxially with CARS excitation light and Raman scattering excitation light;
A Raman scattered light spectrum detecting means for detecting a spectrum of Raman scattered light generated from the sample by irradiating Raman scattering excitation light with the irradiation optical system;
Spectrum analyzing means for analyzing the spectrum of Raman scattered light detected by the Raman scattered light spectrum detecting means and calculating a specific frequency of molecules in the sample;
Detuning setting means for setting the frequency calculated by the spectrum analyzing means as detuning of CARS excitation light;
A laser microscope apparatus comprising: CARS light detection means for detecting CARS light generated from the specimen by irradiating the detuned CARS excitation light set by the detuning setting means with the irradiation optical system. A Raman scattered light spectrum storage unit for storing a spectrum of Raman scattered light detected by the Raman scattered light spectrum detecting means;
2. The laser microscope apparatus according to claim 1, wherein the detuning setting unit sets, as detuning of the CARS excitation light, a frequency at which a spectrum of Raman scattered light stored in the Raman scattered light spectrum storage unit changes with time.   2. The laser microscope apparatus according to claim 1, wherein the detuning setting unit sets a frequency at which the spectrum intensity of Raman scattered light reaches a peak as the detuning of the CARS excitation light.   3. The laser microscope apparatus according to claim 2, wherein the detuning setting unit sets, as detuning of the CARS excitation light, a frequency at which the peak of the spectrum of Raman scattered light exceeds a predetermined threshold. The Raman scattered light spectrum detecting means is
A spectroscopic unit for spectrally separating Raman scattered light generated from the specimen;
2. The laser microscope apparatus according to claim 1, further comprising: a multi-channel detector in which a plurality of channels each detecting a spectrum of Raman scattered light dispersed by the spectroscopic unit are arranged.   The laser microscope apparatus according to claim 1, wherein the irradiation optical system irradiates Raman scattering excitation light with a smaller number of irradiation points than CARS excitation light. A lens for modulating the wavefront of the excitation light source on the optical path of the Raman scattering excitation light source;
The laser microscope apparatus according to claim 1, wherein the surface of the specimen is illuminated by the irradiation optical system. The Raman scattered light spectrum detecting means is a fiber spectroscope including a light receiving unit using an optical fiber and a spectroscopic unit that splits light received by the optical fiber,
The laser microscope apparatus according to claim 7, wherein the light receiving unit is disposed at a position conjugate with the pupil of the objective lens.   The laser microscope apparatus according to claim 1, further comprising time-lapse observation means for performing time-lapse observation. The laser microscope apparatus according to claim 1, further comprising an image acquisition unit that acquires a preview image for designating an observation position.

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