Abstract:
A laser microscope according to the present invention is characterized by comprising a laser light source configured to emit an ultra-short pulse laser beam, a storage unit configured to store at least one of dispersion data and chirp amounts of a plurality of optical members inserted in an optical path, a pulse width adjuster configured to adjust a pulse width of the ultra-short pulse laser beam, and a controller configured to control the pulse width adjuster based on at least one of the dispersion data and the chirp amounts of at least one of a laser wavelength of the laser light source and at least one optical member so that the pulse width is shortened on a sample surface.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-301443, filed Sep. 29, 2000, the entire contents of which are incorporated herein by reference. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a scanning multi-photon excitation laser microscope which detects chemical reaction or fluorescence by multi-photon absorption of a sample and a laser pulse width control method for minimizing a pulse width of a laser beam on a sample surface. 
     2. Description of the Background Art 
     A multi-photon exciting scanning type laser microscope has heretofore been known as a laser microscope, in which a sample as an observation object is irradiated with an ultra-short pulse laser beam, and which detects chemical reaction or fluorescence by multi-photon absorption of the sample. 
     A multi-photon excitation phenomenon occurs at a probability which is proportional to n-power (n=2 for 2-photon excitation, n=3 for 3-photon excitation) of a photon density per unit area and unit time. Therefore, a laser beam of an ultra-short pulse usually of a sub picosecond order is used in a light source for a multi-photon excitation method. 
     However, the laser beam of the sub picosecond pulse does not have a completely single wavelength, and has a wavelength width having correlation with a pulse width. In general, when light passes through an optical system, a speed thereof in a medium is low with a shorter wavelength, and high with a longer wavelength. Therefore, when the pulse laser beam has the wavelength width as described above, a difference is generated in a transmission time in accordance with the wavelength during transmission through the optical system. As a result, so-called chirp occurs in which the pulse width after the transmission through the optical system is expanded as compared with the pulse width before incidence upon the optical system. 
     The multi-photon excitation phenomenon depends on a photon density. Therefore, expansion of the pulse width on a sample surface caused by the chirp deteriorates the probability at which the multi-photon excitation phenomenon occurs. As a result, obtained fluorescence is darkened. 
     It is known that pulse width adjuster, that is, so-called pre-chirp compensation is used as means for solving the problem. The pre-chirp compensation is an optical system configured by a prism pair, a grating pair, or a combination of these pairs. In the pre-chirp compensation, when a long wavelength light of pulse laser is incident behind a short wavelength light, the expansion of the pulse width after the transmission through the optical system is corrected. 
     On the other hand, a case in which the multi-photon excitation method is used in the scanning laser microscope. The scanning laser microscope usually includes one selected from a plurality of objective lenses and other optical systems (such as prism and mirror). These plurality of objective lenses and other optical systems differ from one another in optical path length and material. Therefore, the expansion of the pulse width differs with the selected optical system such as the objective lens. In order to perform the multi-photon excitation method on an optimum condition, it is necessary to adjust the pre-chirp compensation, every time the optical system such as the objective lens is selected. 
     The expansion of the pulse width also depends on the pulse width of the pulse laser beam. Therefore, when the wavelength of the pulse laser beam is variable, the pre-chirp compensation needs to be similarly adjusted for every change of the wavelength. 
     Methods disclosed in Jpn. Pat. Appln. KOKAI Publication No. 10-318924 and the Related U.S. patent application Ser. No. 09/265,183 have heretofore been known as a method for optimizing the adjustment of the pre-chirp compensation. 
     Among these, the Jpn. Pat. Appln. KOKAI Publication No. 10-318924 discloses a method of: disposing an apparatus comprising a collimating optical system disposed opposite to the objective lens via the sample and an autocorrelator for measuring the pulse width; and adjusting the pre-chirp compensation while the pulse width on the sample is measured. 
     Additionally, in the method, since the collimating optical system and autocorrelator are disposed in the vicinity of the sample, the apparatus is enlarged in size. Therefore, for example, patch clamping is performed by inserting an electrode into the sample during observation. In this case, a problem occurs that it is difficult to secure an operation space around the sample. 
     Moreover, the Related U.S. patent application Ser. No. 09/265,183 discloses a method of disposing a correction optical member in accordance with the optical path length of each objective lens, and selecting and disposing the correction optical member in the optical path in accordance with the selected objective lens. 
     In the method, when there are other selected optical systems such as the prism and mirror in addition to the objective lenses, the correction optical members are necessary for the optical systems, and the apparatus therefore increases in size. Moreover, it is also difficult to prepare a large number of correction optical members for all the optical systems. Furthermore, the correction optical members are only changed, and fine adjustment cannot be performed. Therefore, when the laser beam with a variable wavelength is used, it is also difficult to optimize/adjust the pre-chirp compensation in all wavelength areas. 
     BRIEF SUMMARY OF THE INVENTION 
     An object of the present invention is to provide a laser microscope in which an optimum pulse width adjustment can be performed in order to reduce or preferably minimize a pulse width of a laser beam on a sample position. 
     A laser microscope according to the present invention is characterized by comprising: a laser light source configured to emit an ultra-short pulse laser beam; a storage unit configured to store at least one of dispersion data and chirp amounts of a plurality of optical members inserted in an optical path; a pulse width adjuster configured to adjust a pulse width of the ultra-short pulse laser beam; and a controller configured to control the pulse width adjuster based on at least one of the dispersion data and the chirp amounts of at least one of a laser wavelength of the laser light source and at least one optical member so that the pulse width is shortened on a sample surface. 
     Another laser microscope according to the present invention is characterized by comprising: a laser light source configured to emit an ultra-short pulse laser beam; a pulse width adjuster to adjust a pulse width of the ultra-short pulse laser beam; an optical member, attachably/detachably disposed with respect to an optical path, configured to lead the laser beam to a sample; an optical member detector configured to detect attachment/detachment of the optical member with respect to the optical path; a light amount detector configured to detect a light emitted from the sample; and a controller configured to control the pulse width adjuster so as to increase a light amount detected by the light amount detector, when the optical member detector detects the attachment/detachment of the optical member with respect to the optical path. 
     A laser pulse width control method according to the present invention is characterized by comprising: storing at least one of dispersion data and chirp amounts of a plurality of optical members inserted in an optical path; and controlling pulse width adjuster so that the pulse width is shortened on a sample surface based on at least one of the dispersion data and the chirp amounts of at least one optical member corresponding to at least one of a laser wavelength of a laser light source. 
     As a result, according to the present invention, even when a wavelength of ultra-short pulse laser is changed and an optical member such as an objective lens is attached/detached and changed with respect to an optical path, chirp compensation is appropriately adjusted. Adjustment of the pulse width of the ultra-short pulse laser is always controlled so that the laser pulse width on a sample surface is minimized. Therefore, the sample can be observed on an optimum condition. 
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING 
     FIG. 1 shows a schematic configuration of a laser according to the first embodiment of the present invention; 
     FIG. 2 shows a schematic configuration of a laser according to the second embodiment of the present invention; 
     FIG. 3 shows a schematic configuration of a laser according to the third embodiment of the present invention; 
     FIG. 4 shows a schematic configuration of a laser according to the fourth embodiment of the present invention; 
     FIG. 5 shows a schematic configuration of a laser according to the fifth embodiment of the present invention; and 
     FIG. 6 shows a schematic configuration of a laser according to the sixth embodiment of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Embodiments of the present invention will be described hereinafter with reference to the drawings. 
     FIG. 1 shows a schematic configuration of a scanning multi-photon excitation laser microscope to which the present invention is applied. In FIG. 1, an ultra-short pulse laser beam emitted from an ultra-short pulse laser light source  1  which generates the ultra-short pulse laser beam is shaped into a parallel beam by a collimating lens  2 , incident upon a scanner unit  3  via a pre-chirp compensation adjustment apparatus  14  described later, two-dimensionally deflected by a galvano-mirror  5  and incident upon a microscope main body  9  through a projection lens  8 . The laser beam incident upon the microscope main body  9  is deflected along a microscope light axis by a deflection member  10 , and transmitted through a tube lens  11  and objective lens  12  to form an image on a sample  13 . 
     In the sample  13  a multi-photon excitation phenomenon occurs in a focus of the objective lens  12  and fluorescence is emitted. The fluorescence is again taken into the objective lens  12 , and passed through the tube lens  11  and projection lens  8 . Thereafter, the fluorescence is returned to a fixed beam by the galvano-mirror  5 , and separated from the laser beam by a dichroic mirror  4 . Subsequently, the fluorescence is projected into a photomultiplier  7  as a light receiving element by a projection lens  6 , received, processed by a computer (not shown), and constructed as the image. 
     The objective lens  12  of the microscope main body  9  includes a plurality of objective lenses  12   a  to  12   c  which are different from one another in magnification. The objective lenses  12   a  to  12   c  are disposed in a revolver  15  and can selectively be changed on an optical path. Moreover, a differential interference prism  16  is attachably/detachably disposed in the optical path between the tube lens  11  and the objective lens  12 . 
     Respective flags  17   a  to  17   c  are disposed on the objective lenses  12   a  to  12   c . The revolver  15  includes a sensor  18  which detects the flags  17   a  to  17   c  to recognize the objective lenses  12   a  to  12   c  positioned in the optical path. Moreover, a flag  19  is also disposed in the differential interference prism  16 . The microscope main body  9  includes a sensor  20  which detects the flag  19  to recognize the differential interference prism  16  positioned in the optical path. 
     Furthermore, the sensors  18  and  20  are connected to a controller  21 . The controller  21  is connected to a storage apparatus  22  and the pre-chirp compensation adjustment apparatus  14 . 
     The storage apparatus  22  stores respective chirp amounts of the objective lenses  12   a  to  12   c  and differential interference prism  16  with respect to the wavelength of the pulse laser beam of the ultra-short pulse laser light source  1 , for example, as a chirp amount data file  22   a.    
     The controller  21  recognizes types of the objective lenses  12   a  to  12   c  in the optical path and judges whether or not the differential interference prism  16  exists from detection outputs of the sensors  18  and  20 . Moreover, the controller  21  reads the chirp amounts of the corresponding objective lenses  12   a  to  12   c  and differential interference prism  16  from the chirp amount data file  22   a  of the storage apparatus  22  based on a recognition result. Furthermore, the controller  21  obtains the chirp amount to be corrected, obtains driving amount of the driving device  141  of the pre-chirp compensation adjustment apparatus  14  for the chirp amount to be corrected, and outputs the amount to the pre-chirp compensation adjustment apparatus  14 . 
     The pre-chirp compensation adjustment apparatus  14  is configured of a prism pair, a grating pair, or a combination of these pairs as a known technique. In the present invention, when the pre-chirp compensation adjustment apparatus  14  is driven by a driving device  141 , the chirp amount can be adjusted. In the present invention, the chirp amounts of fixed optical members positioned beforehand on the optical path, such as the dichroic mirror  4 , projection lens  8  and tube lens  11  are set as initial adjustment amounts. Moreover, the driving device  141  adjusts the pre-chirp compensation by the chirp amount to be corrected which is given to the initial adjustment amount by the controller  21 . An expansion of a pulse width of the ultra-short pulse laser beam is thus adjusted. 
     In the aforementioned configuration, when the ultra-short pulse laser light source  1  emits the ultra-short pulse laser beam, the laser beam is passed through the objective lens  12  and focused on the sample  13  similarly as described above. The laser beam causes multi-photon excitation phenomenon in the focus of the objective lens  12  and fluorescence is emitted. The fluorescence is again taken into the objective lens  12 , and received by the photomultiplier  7  via the dichroic mirror  4  and projection lens  6 . The sample  13  is observed from the image constructed by the computer. 
     To change an observation condition from this state, the revolver  15  changes the objective lenses  12   a  to  12   c  positioned on the optical path, the objective lens  12   b  is positioned on the optical path as shown in FIG. 1, and the differential interference prism  16  is additionally inserted into the optical path. The objective lens  12   b  is recognized by the sensor  18 , the differential interference prism  16  is recognized by the sensor  20 , and these detection outputs are sent to the controller  21 . 
     The controller  21  recognizes the objective lens  12   b  and differential interference prism  16  from the detection outputs of the sensors  18  and  20 , and reads the chirp amounts of the corresponding objective lens  12   b  and differential interference prism  16  from the chirp amount data file  22   a  of the storage apparatus  22 . Furthermore, the controller  21  obtains the chirp amount to be corrected, and outputs correction information to the pre-chirp compensation adjustment apparatus  14 . 
     The pre-chirp compensation adjustment apparatus  14  adjusts the pre-chirp compensation via the driving device  141  by the chirp amount to be corrected with respect to the predetermined initial adjustment amount. Thereby, the expansion of the pulse width of the ultra-short pulse laser beam is adjusted, so that the pulse width of the pulse laser beam on the sample  13  is reduced or preferably minimized. 
     Therefore, in this case, when the objective lenses  12   a  to  12   c  are selectively changed on the optical path, and the differential interference prism  16  is attached/detached with respect to the optical path, changes of the chirp amounts are automatically corrected, and the chirp compensation is appropriately adjusted. Therefore, since the pulse width of the laser beam on the sample  13  can always be kept to be short or preferably minimum, the sample can be observed on the optimum condition. 
     FIG. 2 is a figure showing a schematic configuration according to the second embodiment of the present invention. The same part as that of FIG. 1 is denoted with the same reference numerals. 
     An ultra-short pulse laser light source  31  having a wavelength change mechanism  32  can arbitrarily change the wavelength of the ultra-short pulse laser beam, changes the wavelength in accordance with absorption wavelength of a fluorescent dyestuff for use in observation, and emits the ultra-short pulse laser beam. 
     A mirror  33  is disposed midway in the optical path of the ultra-short pulse laser beam emitted by the ultra-short pulse laser light source  31 . A part of the ultra-short pulse laser beam is branched by the mirror  33 , and incident upon a spectrum analyzer  35  as wavelength recognition means via a spectrum analyzer head  34 . The spectrum analyzer  35  recognizes the wavelength of the ultra-short pulse laser beam emitted by the ultra-short pulse laser light source  31 . 
     The storage apparatus  22  includes a first storage section  22   b  and a second storage section  22   c . The first storage section  22   b  stores dispersion data of respective optical members positioned in the optical path, such as dichroic mirror  4 , projection lens  8 , tube lens  11 , and objective lens  12  in the example shown in FIG.  2 . The second storage section  22   c  stores a conversion equation which converts the dispersion data, and the like to the chirp amount. Furthermore, a laser wavelength, and dispersion data of the optical member inserted in the observation optical system are used as parameters of the conversion equation. Additionally, a chirp correction amount of a pre-chirp compensator is obtained by the conversion equation. The controller  21  obtains the chirp amount by calculation based on the wavelength of the ultra-short pulse laser beam recognized by the spectrum analyzer  35  and the dispersion data of the respective optical members stored in the storage apparatus  22 , and outputs a calculation result to the pre-chirp compensation adjustment apparatus  14 . The chirp amount of each optical member on the optical path with respect to the wavelength of the ultra-short pulse laser which becomes a reference is preset as the initial adjustment value in the pre-chirp compensation adjustment apparatus  14 . The pre-chirp compensation adjustment apparatus  14  obtains a difference between the initial adjustment value and the chirp amount given by the controller  21 . Moreover, when the pre-chirp compensation adjustment apparatus  14  adjusts the pre-chirp compensation via the driving device  141  in accordance with the difference, the expansion of the pulse width of the ultra-short pulse laser beam is adjusted. 
     Even when the wavelength of the ultra-short pulse laser beam of the ultra-short pulse laser light source  31  is changed, the change of the chirp amount is automatically corrected, and the chirp compensation is appropriately adjusted. Therefore, since the pulse width of laser on the sample  13  can always be kept to be short or preferably minimum, the sample can be observed on the optimum condition. 
     FIG. 3 is a figure showing a schematic configuration according to the third embodiment of the present invention. The same part as that of FIGS. 1 and 2 is denoted with the same reference numerals. 
     In the third embodiment, similarly as the first embodiment, the plurality of objective lenses  12   a  to  12   c  are changeably disposed in the revolver  15 , and the differential interference prism  16  is attachably/detachably disposed with respect to the optical path. Furthermore, the respective flags  17   a  to  17   c  are disposed in the objective lenses  12   a  to  12   c , and the sensor  18  which detects the flags  17   a  to  17   c  is disposed in the revolver  15 , so that the changeover of the respective objective lenses  12   a  to  12   c  can be detected. Moreover, the flag  19  is disposed in the differential interference prism  16 , and the sensor  20  which detects the flag  19  is disposed in the microscope main body  9 , so that attachment/detachment of the differential interference prism  16  with respect to the optical path can be detected. 
     On the other hand, similarly as the second embodiment, the wavelength of the ultra-short pulse laser light source  31  can be changed by the wavelength change mechanism  32 , a part of the pulse laser beam having the changed wavelength is incident upon the spectrum analyzer head  34  via the mirror  33 , and the wavelength is recognized by the spectrum analyzer  35 . 
     Similarly as the second embodiment, the storage apparatus  22  stores the dispersion data of the objective lenses  12   a  to  12   c  and differential interference prism  16 . The controller  21  obtains the chirp amount by calculation from the wavelength of the ultra-short pulse laser beam recognized by the spectrum analyzer  35  and the dispersion data stored in the storage apparatus  22  in accordance with the objective lenses  12   a  to  12   c  and differential interference prism  16  positioned on the optical path, and outputs the chirp amount as correction information to the pre-chirp compensation adjustment apparatus  14 . The chirp amount of the fixed optical member on the optical path with respect to the wavelength of the ultra-short pulse laser beam as the reference is preset as the initial adjustment value in the pre-chirp compensation adjustment apparatus  14 . The pre-chirp compensation adjustment apparatus  14  adjusts the pre-chirp compensation via the driving device  141  with respect to the initial adjustment value only by the chirp amount to be corrected, and adjusts the expansion of the pulse width of the ultra-short pulse laser beam. 
     According to the third embodiment, even with the changeover of the objective lenses  12   a  to  12   c  with respect to the optical path, attachment/detachment of the differential interference prism  16 , and the change of wavelength of the ultra-short pulse laser beam of the ultra-short pulse laser light source  31 , the chirp amount is automatically corrected. As a result, the chirp compensation is appropriately adjusted. Therefore, since the pulse width of laser beam on the sample  13  can always be kept to be short or preferably minimum, the sample can be observed on the optimum condition. 
     FIG. 4 is a figure showing a schematic configuration of a fourth embodiment of the present invention. The same part as that of FIG. 3 is denoted with the same reference numerals. 
     The plurality of objective lenses  12   a  to  12   c  are held by an electromotive revolver  37  having a motor  36 . Moreover, the differential interference prism  16  is disposed to be attachable/detachable by a motor  38 . Furthermore, the ultra-short pulse laser light source  31  has the electromotive wavelength change mechanism  32 . 
     These motors  36 ,  38  and wavelength change mechanism  32  are connected to the controller  21 , and remote control is enabled by an operation device  39  which is input means. The respective motors  36 ,  38  are driven via the controller  21 , and the wavelength change mechanism  32  is controlled in accordance with designated information of the optical member inputted into the operation device  39  and designated information of the laser wavelength. 
     Similarly as the second embodiment, the storage apparatus  22  stores the dispersion data of the objective lenses  12   a  to  12   c , differential interference prism  16 , and the like. The controller  21  obtains the chirp amount by calculation from the dispersion data stored in the storage apparatus  22  in accordance with the wavelength of the ultra-short pulse laser beam and the dispersion data stored in the storage apparatus  22  in accordance with the objective lenses  12   a  to  12   c  and differential interference prism  16  based on the designated information of the optical member from the operation device  39  and the designated information of the laser wavelength. Then, the controller  21  outputs a calculated value as the correction information to the pre-chirp compensation adjustment apparatus  14 . The chirp amount of the fixed optical member on the optical path with respect to the wavelength of the ultra-short pulse laser beam as the reference is preset as the initial adjustment value in the pre-chirp compensation adjustment apparatus  14 . The pre-chirp compensation adjustment apparatus  14  drives the driving device  141  only by the chirp amount to be corrected with respect to the initial adjustment value, adjusts the pre-chirp compensation, and adjusts the expansion of the pulse width of the ultra-short pulse laser beam. 
     According to the fourth embodiment, the changeover of the objective lenses  12   a  to  12   c  with respect to the optical path, attachment/detachment of the differential interference prism  16 , and the change of wavelength of the ultra-short pulse laser beam of the ultra-short pulse laser light source  31  can remotely be operated by the external operation device  39 . Furthermore, the change of the chirp amount generated by the remote operation is automatically corrected, and the chirp compensation is appropriately adjusted. Therefore, since the pulse width of laser beam on the sample  13  can always be kept to be short or preferably minimum, the sample can be observed on the optimum condition. 
     FIG. 5 is a figure showing a schematic configuration according to the fifth embodiment of the present invention. The same part as that of FIG. 1 is denoted with the same reference numerals. 
     The plurality of objective lenses  12   a  to  12   c  are changeably disposed in the revolver  15 , and the differential interference prism  16  is attachably/detachably disposed with respect to the optical path. Respective flags  40   a  to  40   c  are disposed in a position corresponding to an objective lens attachment section in the revolver  15 . The sensor  18  which detects the flags  40   a  to  40   c  is disposed in the revolver  15 , so that a rotating operation of the revolver  15 , that is, the changeover of the respective objective lenses  12   a  to  12   c  can be detected. Moreover, the flag  19  is disposed in the differential interference prism  16 , and the sensor  20  which detects the flag  19  is disposed in the microscope main body  9 , so that attachment/detachment of the differential interference prism  16  with respect to the optical path can be detected. 
     When the sensor  18  detects the changeover of the objective lenses  12   a  to  12   c  by the revolver  15 , or the sensor  20  detects the attachment/detachment of the differential interference prism  16  with respect to the optical path, the controller  21  stores the operation. Subsequently, when the image is acquired, the controller  21  monitors the output of the photomultiplier  7  while driving the driving device  141  of the pre-chirp compensation adjustment apparatus  14 . A position in which the output of the photomultiplier  7  is increased or preferably maximized is searched, and the driving mechanism  141  is stopped in the position. That is, where the output of the photomultiplier  7  increases, the correction of the chirp amount is optimum, and the pulse width on the sample  13  becomes short or preferably minimum. 
     According to the fifth embodiment, for the changeover of the objective lenses  12   a  to  12   c  with respect to the optical path, and the attachment/detachment of the differential interference prism  16 , the chirp amount is automatically corrected. Therefore, since the pulse width of laser beam on the sample  13  can always be kept to be short or preferably minimum, the sample can be observed on the optimum condition. 
     FIG. 6 is a figure showing a schematic configuration of a sixth embodiment of the present invention. The same part as that of FIG. 5 is denoted with the same reference numerals. 
     The plurality of objective lenses  12   a  to  12   c  are held by the electromotive revolver  37  having the motor  36 . Moreover, the differential interference prism  16  is also disposed to be attachable/detachable by the motor  38 . These motors  36 ,  38  are connected to the controller  21 , and can remotely be controlled by the operation device  39 . 
     When an instruction for changing the objective lenses  12   a  to  12   c  by the electromotive revolver  37  or an instruction for attaching/detaching the differential interference prism  16  is inputted into the controller  21  from the operation device  39 , the controller  21  stores the instruction. Subsequently, when the operation for acquiring the image is executed, the controller  21  monitors the output of the photomultiplier  7  while driving the driving device  141  of the pre-chirp compensation adjustment apparatus  14 . The position in which the output of the photomultiplier  7  is increased or preferably maximized is searched, and the driving mechanism  141  is stopped in the position. That is, where the output of the photomultiplier  7  is increased or preferably maximized, the correction of the chirp amount is optimum, and the pulse width on the sample  13  becomes short or preferably minimum. 
     According to the sixth embodiment, the changeover of the objective lenses  12   a  to  12   c  or the attachment/detachment of the differential interference prism  16  with respect to the optical path can remotely be operated by the external operation device  39 . Furthermore, the change of the chirp amount generated by the remote operation is automatically corrected, and the pulse width of laser beam on the sample  13  can always be kept to be short or preferably minimum, so that the sample can be observed on the optimum condition. 
     Additionally, in the aforementioned embodiments, the objective lens  12  and differential interference prism  16  have been described as the attachable/detachable and changeable optical members, but the present invention is not limited to these, and can also similarly be applied to the other optical members such as the lens, mirror, and prism. Moreover, a technique of pre-chirp compensation is not limited to the prism pair described in the shown embodiment, and the present invention is similarly applied to the technique of the grating pair or the combination of these pairs. Furthermore, the ultra-short pulse laser light source  31  whose wavelength is variable is not limited to the source which can continuously vary the wavelength with a single unit. An apparatus for changing and using laser in a system in which a plurality of lasers are controlled by a combiner can also similarly be applied. Moreover, the laser microscope is not limited to the scanning laser fluorescent microscope for observing fluorescence generated by the multi-photon excitation phenomenon. The present invention can similarly be applied also to a laser microscope in which high energy in a pulse peak is utilized to finely process the sample. Furthermore, the present invention is not limited to the aforementioned embodiments, and can variously be changed within the scope of the present invention. For example, the respective embodiments may also be combined with one another. 
     As described above, according to the present invention, the optimum pulse width adjustment can be performed so as to reduce or preferably minimize the pulse width of the laser beam in the sample position. There can therefore be provided the laser microscope in which the sample can be observed under the optimum condition. 
     Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the present invention in its broader aspects is not limited to the specific details, representative devices, and illustrated examples shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.