Abstract:
A laser scanning microscope separates fluorescence signals of different fluorophores in accurate unmixing by eliminating positional pixels shifts between different fluorescence images obtained through irradiation of different-wavelength laser lights. The microscope includes a laser light source capable of emitting a wavelength-changeable laser light, a correction amount determination unit that determines a correction amount for correcting an optical axis shift of the laser light, an optical axis adjusting unit that adjusts an optical axis, a scanning unit that performs two-dimensional scanning, an objective lens that focuses the laser scanning light to a specimen and fluorescence emitted from the specimen, a light detector that detects the fluorescence, and a control unit that changes the wavelength of the laser light synchronously with the scanning by the scanning unit while controlling the optical axis adjusting unit based on the correction amount determined by correction amount determination unit.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to laser scanning microscopes. 
     This application is based on Japanese Patent Application No. 2007-037832, the content of which is incorporated herein by reference. 
     2. Description of Related Art 
     There is a known technique for separating some different fluorescence each of whose wavelength components are overlapping each other, by unmixing processing a set of images obtained through use of a laser light source whose wavelength is changeable, such as a pulsed laser light source (see Japanese Unexamined Patent Application, Publication No. 2004-233351, for example). 
     In the known technique disclosed in Japanese Unexamined Patent Application, Publication No. 2004-233351, however, since a pulsed laser light source whose wavelength is changeable is used, the optical axis of the optical path running from the laser light source to the specimen may be shifted when the wavelength of the laser light is varied. 
     If the optical axis of a laser light is shifted while the wavelength of the laser light is sequentially varied, pixels of each fluorescence image obtained by irradiating laser lights of different wavelengths may also be positionally shifted from each other. Consequently, unmixing processing of such a plurality of fluorescence images whose pixels are positionally shifted from each other results in a meaningless process because pixels of different images have no commonality. Accordingly, spectral unmixing (separating the fluorescence signals of different fluorophores) cannot be achieved. 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention provides a laser microscope that can obtain fluorescence images whose positional shifts between pixels of different fluorescence images obtained through irradiation of laser lights having different wavelengths are eliminated, then accurate unmixing processing to separate the fluorescence signals of different fluorophores can be realized. 
     According to a first aspect of the present invention, a laser scanning microscope includes a laser light source capable of emitting a laser light whose wavelength is changeable, a correction amount determination unit that determines a correction amount for correcting an optical axis shift of the laser light by changing the laser light having the different wavelength, a scanning unit that performs two-dimensional scanning with the laser light emitted from the laser light source, an objective lens that focuses the laser light from the scanning unit to a specimen and collects fluorescence emitted from the specimen, a light detector that detects the fluorescence collected by the objective lens, and a control unit that changes the wavelength of the laser light from the laser light source synchronously with the scanning the laser light performed by the scanning unit while controlling the optical axis adjusting unit on the basis of the correction amount of the optical axis shift determined by the correction amount determination unit. 
     In the first aspect, the scanning unit performs two-dimensional scanning with the laser light emitted from the laser light source, and the laser light is focused by the objective lens to be irradiated to the specimen, whereby a fluorescent material contained in the specimen is excited to emit fluorescence. The emitted fluorescence is collected by the objective lens and is detected as fluorescence intensity information by the light detector. By storing scanning positions of the scanning unit in association with the fluorescence intensity information, a two-dimensional fluorescence image can be obtained. 
     When the wavelength of the laser light is changed, the optical axis of the laser light is shifted. The correction amount determination unit determines a correction amount for correcting an optical axis shift while the optical axis adjusting unit adjusts the optical axis so as to eliminate the shift in the optical axis on the basis of the correction amount. Therefore, even if the wavelength of the laser light is varied, the laser light can be irradiated to the specimen along a constant optical axis. Further, the control unit causes the wavelength of the laser light emitted from the laser light source to change synchronously with the laser light scanning performed by the scanning unit, while controlling the optical axis adjusting unit to eliminate the shift in the optical axis. Therefore, even if the wavelength of the laser light is varied, fluorescence images with the same optical axis can be obtained. Consequently, positional pixel shifts between fluorescence images obtained through. irradiation of laser lights of different wavelengths can be eliminated, whereby accurate unmixing can be performed. 
     The laser scanning microscope according to the first aspect may further include an optical axis shift detecting unit that detects the amount of an optical axis shift by varying the laser light having the different wavelength, the correction amount determination unit may determine the correction amount on the basis of the detected amount of the optical axis shift. 
     The laser scanning microscope according to the first aspect may further include a light modulating unit that adjusts intensity of the laser light, a storage unit that stores wavelength information of the laser light in association with intensity information of the laser light, and a light modulating control unit that controls the light modulating unit in accordance with the intensity information of the laser light stored in the storage unit when the wavelength of the laser light emitted from the laser light source is changed. 
     When the wavelength of the laser light emitted from the laser light source is varied, the intensity of the laser light emitted from the laser light source varies. 
     With the above-described configuration, when the wavelength of the laser light emitted from the laser light source is changed, the light modulating control unit controls the light modulating unit in accordance with the intensity information stored in the storage unit in association with the wavelength information. Therefore, a laser light with a constant intensity can be irradiated whether or not the wavelength is changed. Consequently, variations in the intensity of fluorescence images obtained for different wavelengths can be suppressed. 
     The laser scanning microscope according to the first aspect may further include a light modulating unit that adjusts intensity of the laser light, a power monitor that detects the intensity of the laser light, and a light modulating control unit that controls the light modulating unit such that the intensity detected by the power monitor is maintained at a constant level when the wavelength of the laser light emitted from the laser light source is changed. 
     With such a configuration, the light modulating control unit controls the light modulating unit such that the intensity detected by the power monitor is maintained at a constant level. Therefore, even if the wavelength of the laser light emitted from the laser light source is changed, a laser light with a constant intensity can be irradiated. Consequently, variations in the intensity of fluorescence images obtained for different wavelengths can be suppressed. 
     The laser scanning microscope according to the first aspect may further include a sensitivity control unit that adjusts the sensitivity of the light detector in accordance with the wavelength of the laser light emitted from the laser light source. 
     With such a configuration, when the intensity of the laser light emitted from the laser light source changes with the wavelength thereof, the sensitivity control unit adjusts the detection sensitivity of the light detector. Therefore, even if the wavelength is changed, fluorescence images with a constant intensity can be obtained. 
     The laser scanning microscope according to the first aspect may further include a storage unit that stores, in association with the wavelength of the laser light emitted from the laser light source, an adjustment value for the optical axis adjusting unit controlled in response to change of the wavelength. 
     With such a configuration, an adjustment value for the optical axis adjusting unit is stored in the storage unit in association with the wavelength for each change of the wavelength. Therefore, when the same wavelength is selected afterward, the adjustment value stored in the storage unit is used to realize quick optical axis adjustment using the optical axis adjusting unit, without requiring the optical axis shift detecting unit to perform detection and the control unit to identify the adjustment value for the optical axis adjusting unit. 
     In the laser scanning microscope according to the first aspect, the scanning unit may include a driving mechanism that moves a scanning plane of the laser light in a direction of the optical axis relative to the specimen, and the control unit may operate to change the wavelength of the laser light synchronously with three-dimensional scanning in which the driving mechanism moves the scanning plane in the direction of the optical axis while two-dimensional scanning with the laser light is performed along a focal plane of the objective lens. 
     With such a configuration, the number of changing operation of the laser light wavelength can be minimized, whereby a three-dimensional fluorescence image with no positional pixel shifts can be obtained in a shorter time through irradiation of laser lights with different wavelengths. 
     According to a second aspect of the invention, a laser scanning microscope includes a laser light source capable of emitting a laser light whose wavelength is changeable, a storage unit that stores the wavelength of the laser light from the laser light source in association with a correction amount of an optical axis shift, an optical axis adjusting unit that adjusts the optical axis of the laser light emitted from the laser light source, a scanning unit that performs two-dimensional scanning with the laser light emitted from the laser light source, an objective lens that focuses the laser light from the scanning unit to a specimen and collects fluorescence emitted from the specimen, a light detector that detects the fluorescence collected by the objective lens, and a control unit that changes the wavelength of the laser light from the laser light source synchronously with the scanning with the laser light performed by the scanning unit while controlling the optical axis adjusting unit on the basis of the correction amount of the optical axis shift read from the storage unit. 
     According to the invention, separating the fluorescence signals of different fluorophores can be achieved in accurate unmixing processing realized by eliminating positional shifts between pixels of different fluorescence images obtained through irradiation of laser lights having different wavelengths. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIG. 1  is a block diagram showing the entire configuration of a laser scanning microscope according to an embodiment of the invention. 
         FIG. 2  is a flowchart showing the process of observation of a specimen through the laser scanning microscope shown in  FIG. 1 . 
         FIG. 3  is a graph showing a wavelength characteristic with respect to the intensity of a laser light emitted from a laser light source of the laser scanning microscope shown in  FIG. 1 . 
         FIG. 4  is a flowchart showing the process of observation of a specimen through a modification of the laser scanning microscope shown in  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A laser scanning microscope  1  according to an embodiment of the present invention will now be described with reference to  FIGS. 1 to 3 . 
     Referring to  FIG. 1 , the laser scanning microscope  1  according to the embodiment includes the following: an illumination optical system  3  that irradiates an ultrashort-pulse laser light (hereinafter referred to as a laser light) to a specimen A placed on a stage  2 , an observation optical system  4  that detects fluorescence emitted from the specimen A, a control device  5  that controls the illumination optical system  3  and the observation optical system  4 , an input device  6  for inputting various settings, and a display device  7  that displays an obtained fluorescence image. 
     The illumination optical system  3  includes the following: a laser light source  8  that can emit a multi-wavelength laser light; an acoustooptic element (light modulating unit)  9 , such as an acoustooptic tunable filter (AOTF), that turns the laser light emitted from the laser light source  8  on or off, controls the emitted laser light, and selects the wavelength of the laser light; a negative-chirp (NC) unit  10  that preferentially transmits shorter-wavelength light; an optical axis adjusting unit  11  that can adjust the offset and inclination of the optical axis of the laser light; an optical axis shift detecting unit  12  that detects a shift in the optical axis of the laser light; a power monitor  13  that detects the intensity of the laser light; a scanner  14  that performs two-dimensional scanning with the laser light; and an objective lens  15  that irradiates the laser light from the scanner  14  to the specimen A and collects fluorescence emitted from the specimen A. 
     The illumination optical system  3  further includes a beam expander  16  that changes the focal plane of the laser light emitted through the objective lens  15  in the optical axis direction, and a shutter  17  that permits or blocks entry of the laser light into the scanner  14 . In  FIG. 1 , a reference numeral  18  denotes a mirror. 
     The optical axis adjusting unit  11  is a combination of a motor and two flat mirrors (not shown). When the two flat mirrors are simultaneously moved in parallel, the optical axis of the laser light can be offset in a direction orthogonal thereto. When the flat mirrors are rotated about an axis orthogonal to the optical axis, the inclination angle of the laser light can be adjusted. 
     The optical axis shift detecting unit  12  includes, for example, a beam splitter  19  that separates a portion of the laser light from the optical path on the downstream side of the beam expander  16 , and two sensors  20  and  21  that detect, via different optical path lengths, the portion of the laser light separated from the optical path by the beam splitter  19 . Reference numerals  22  and  23  denote a beam splitter and a mirror, respectively. 
     Each of the sensors  20  and  21 , which are sensors such as four-segment photodiodes, are configured to detect the amount of offset of the optical axis of the laser light in a direction orthogonal to the original optical axis on the basis of the output balance between four sensor segments (not shown) provided in correspondence with spot positions at which the laser light is received. In accordance with the difference between the amount of offsets detected by the two sensors  20  and  21  positioned via different optical path lengths, the inclination angle with respect to the original optical axis of the laser light is detected. 
     The scanner  14  includes, for example, two galvanometer mirrors  14   a  and  14   b  positioned opposite to each other and supported in such a manner that can be rocked back and forth about respective axes in two mutually orthogonal directions. With the scanner  14 , the two galvanometer mirrors  14   a  and  14   b  are rocked synchronously, whereby a scanning operation with the laser light, such as two-dimensional raster scanning, can be performed. 
     The power monitor  13  includes, for example, a beam splitter  24  that separates a portion of the laser light from the optical path on the downstream side of the optical axis shift detecting unit  12 ; a diaphragm  25 , which is the equivalent of an objective pupil, that transmits the portion of the laser light separated from the optical path by the beam splitter  24 ; and a sensor  26  that detects the laser light that has passed through the diaphragm  25 . 
     The observation optical system  4  includes a dichroic mirror  27  that separates fluorescence collected by the objective lens  15  from the optical path of the laser light between the objective lens  15  and the scanner  14 ; a barrier filter  28  that removes the laser light from the fluorescence separated by the dichroic mirror  27 ; and a light detector  29 , such as a photomultiplier, that detects the fluorescence transmitted through the barrier filter  28 . 
     The control device  5  also works as a correction amount determination unit that determines a correction amount for correcting an optical axis shift of the laser light by changing the laser light having the different wavelength. 
     The control device  5  outputs a command signal for selecting a laser light wavelength to the acoustooptic element  9 . Further, the control device  5  receives a detection signal from the optical axis shift detecting unit  12 , calculates an adjustment value for the optical axis adjusting unit  11 , and outputs the adjustment value to the optical axis adjusting unit  11 . Further, the control device  5  outputs a command signal for adjusting the intensity of the laser light to the acoustooptic element  9  in accordance with the laser light intensity detected by the power monitor  13 . 
     Further, the control device  5  notifies the negative chirp unit  10  of a chirp amount according to the laser light wavelength, and instructs the beam expander  16  to adjust the laser light diameter in accordance with the laser light wavelength. 
     Further, the control device  5  turns the shutter  17  to the off state so as to block the entry of the laser light into the scanner  14  when varying the laser light wavelength, and turns the shutter  17  to the on state so as to permit the irradiation of the laser light to the specimen A after completing the above-described adjustment operations. 
     Further, the control device  5  constructs a fluorescence image on the basis of information on the brightness of the fluorescence detected by the light detector  29  and information on the scanning position of the scanner  14 , and outputs the image to the display device  7 . 
     Operation of the laser scanning microscope  1  according to the embodiment having the above-described configuration will now be described. 
       FIG. 2  shows the process of fluorescence observation of the specimen A through the laser scanning microscope  1  according to the embodiment. In step S 1 , observation conditions are input to the control device  5  through the input device  6 . This embodiment concerns the case where the wavelength of the laser light emitted from the laser light source  8  is changed, whereby a two-dimensional fluorescence image formed along the focal plane of the objective lens  15  is obtained for each wavelength. 
     The control device  5  controls necessary components in accordance with the input observation conditions. The observation conditions include desired laser light intensity and the number of laser light scans. 
     In step S 2 , the control device  5  outputs an off command signal to the shutter  17  so as to block the optical path running from the laser light source  8  toward the scanner  14 . In this state, in step S 3 , the control device  5  causes the laser light source  8  to emit a laser. The laser light contains multi-wavelength light. Therefore, in step S 4 , the control device  5  inputs a wavelength selection command signal to the acoustooptic element  9  to cause the acoustooptic element  9  to emit a laser light having the selected wavelength. 
     Since a wavelength of the laser light has been selected, the optical axis and intensity of the laser light to be emitted from the acoustooptic element  9  varies. In response to this, in step S 5 , the optical axis shift detecting unit  12  detects the amount of offset and inclination angle of the optical axis. Then, in step S 6 , the control device  5  calculates an adjustment value for the optical axis adjusting unit  11  on the basis of the detection signals, thereby controlling the optical axis adjusting unit  11 . Thus, a shift in the optical axis is eliminated at the optical axis shift detecting unit  12 . In step S 7 , the power monitor  13  detects the laser light intensity. In step S 8 , on the basis of the intensity signal, the control device  5  commands the acoustooptic element  9  to adjust the intensity to the desired intensity, whereby the laser light is adjusted to a predetermined intensity. 
     In step S 9 , after the completion of the optical axis adjustment and intensity adjustment, the control device  5  outputs an on command signal to the shutter  17 , whereby the laser light emitted from the laser light source  8  is made to output toward the scanner  14 . 
     In step S 10 , the laser light enters the scanner  14  for two-dimensional scanning, and is collected by the objective lens  15  to be irradiated to the specimen A on the stage  2 . 
     The irradiated laser light excites a fluorescent material contained in the specimen A, whereby fluorescence is emitted. Since an ultrashort-pulse laser light is used, multiphoton fluorescence is emitted in an extremely thin region along the focal plane of the objective lens  15  as a result of multiphoton excitation. 
     The emitted fluorescence is collected by the objective lens  15 , is separated on the way back along the same optical path by the dichroic mirror  27 , and is detected by the light detector  29  through the barrier filter  28 . 
     Information on the intensity of the fluorescence detected by the light detector  29  and information on the scanning position of the scanner  14  at each moment of fluorescence detection are sent to the control device  5 . The control device  5  associates and stores the two kinds of information. The scanner  14  performs raster scanning of a predetermined imaging region with the laser light, whereby a two-dimensional multiphoton fluorescence image is constructed and displayed on the display device  7 . 
     In step S 11  of the embodiment, when the laser light scanning of the predetermined imaging region is completed, whether or not the number of scans is equal to a preset number of scans is checked. If the preset number of scans is not reached, the process returns to step S 2  to be repeated therefrom. If the preset number of scans is reached, the process ends. 
     With the laser scanning microscope  1  according to the embodiment, the laser light is maintained with a certain wavelength during a single scan for obtaining a multiphoton fluorescence image of a predetermined imaging region of the specimen A. Synchronously with the completion of each scan, the wavelength of the laser light is changed. For each change, the optical axis shift and the laser light intensity are corrected. Therefore, problems of positional pixel shifts and nonuniform fluorescence intensities between different fluorescence images obtained through irradiation of laser lights having different wavelengths can be eliminated. 
     Accordingly, an advantage is afforded in that a linear. regression analysis using a plurality of fluorescence images obtained as above can realize accurate unmixing and separation of fluorescence components emitted with overlapping wavelengths through irradiation of laser lights having different wavelengths. 
     The laser scanning microscope  1  according to the embodiment is explained as an example of a multiphoton-excitation type using an ultrashort-pulse laser light. However, the microscope is not limited thereto, and may also be employed a confocal microscope using a continuous-wave laser light. 
     In the embodiment, the intensity of a laser light with any wavelength is detected by the power monitor  13  and is adjusted by the acoustooptic element  9  to obtain desired intensity. Instead of this, by providing a look-up table that stores associations between wavelengths and intensities of the laser light, the laser light intensity may be maintained at a constant level without the power monitor  13 . Referring to  FIG. 3 , the laser light intensity varies in response to the laser light wavelength. Accordingly, with reference to a look-up table prepared on the basis of the relationship shown in  FIG. 3 , a command signal to be sent to the acoustooptic element  9  may be set to such a value as to maintain the laser light intensity at a constant level regardless of the laser light wavelength. 
     In the embodiment, the acoustooptic element  9  is used to turn the laser light on or off, adjust the laser light intensity, and select the laser light wavelength. Instead of this, a modulator of another type, such as an electrooptic element, may be used. 
     In the embodiment, the acoustooptic element  9  is used to adjust the laser light so as to maintain the intensity of the laser light to be irradiated to the specimen A at a constant level regardless of the laser light wavelength. Instead of this, the obtained fluorescence intensity may be adjusted instead of adjusting the laser light intensity. In other words, irradiation of a laser light having a wavelength characteristic shown in  FIG. 3  to the specimen A causes the specimen A to emit fluorescence having intensity to be proportional to the irradiated intensity of the laser light. Accordingly, by adjusting the light-receiving sensitivity of the light detector  29  in accordance with a wavelength characteristic to be inversely proportional to the wavelength characteristic shown in  FIG. 3 , fluorescence images for which relative relationships of fluorescence intensities thereof are corrected independently from laser light wavelengths can be obtained. 
     A storage unit may also be provided for storing adjustment values set for the optical axis adjusting unit  11  and intensity adjustment values set for the acoustooptic element  9  every time the laser light wavelength is varied. With such a storage unit, when a laser light of the same wavelength is irradiated, adjustment values stored in the storage unit can be used. Consequently, the execution of alignment adjustment and power monitoring become unnecessary again, whereby observation time can be reduced. 
     In the embodiment, a multiphoton fluorescence image formed along a single focal plane of the objective lens  15  is obtained by changing in the laser light wavelength. In addition to this, a driving mechanism  30 , as shown in  FIG. 1 , that moves the focal plane of the objective lens  15  in the optical axis direction during scanning may be provided. In that case, a plurality of two-dimensional multiphoton fluorescence images can be obtained along different focal planes of the objective lens  15 , the focal planes being parallel to each other at intervals in the optical axis direction. If the interval is set to a sufficiently small, a three-dimensional multiphoton fluorescence image of the specimen A can be obtained. 
     In that case, by setting the laser light wavelength to change for every scan in the optical axis direction, the number of wavelength changing processes can be minimized, and a plurality of three-dimensional multiphoton fluorescence images resulting from irradiation of laser lights having different wavelengths can be obtained quickly. 
     In the embodiment, the control device  5  receives the detection signal from the optical axis shift detecting unit  12  and calculates an adjustment value for the optical axis adjusting unit  11 . Instead, a storage unit  31 , as shown in  FIG. 1 , connected to the control device  5  may be provided for storing all wavelengths selected by the acoustooptic element  9  in association with the corresponding adjustment values (the correction amount of the optical axis shift) for the optical axis adjusting unit  11 . In that case, referring to step S 5 ′in  FIG. 4 , when the control device  5  selects a laser light wavelength through the acoustooptic element  9 , the control device  5  may read the corresponding adjustment value in response to the selected wavelength stored in the storage unit, thereby controlling the optical axis adjusting unit  11 . 
     In the embodiment, when the wavelength is changed or the alignment is adjusted, the shutter  17  blocks the laser light to prevent the specimen A from being irradiated with the laser light before the laser light is stabilized. However, the invention is not limited thereto. The shutter  17  may not necessarily be provided.