Patent Abstract:
There is disclosed a laser scanning microscope including a first optical scanning system which scans a first laser light for observing a sample on the sample, a first light branch device which branches the light from the sample from an optical path of the first laser light, a photodetector which detects the light from the sample, separated by the first light branch device, a second optical scanning system which irradiates a specific portion on the sample with a second laser light for stimulating or operating the sample, and a wavelength selection device which is disposed between the first light branch device and photodetector and which includes a first function of transmitting a desired observation light and a second function of limiting transmission of the second laser light.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2002-250824, filed Aug. 29, 2002, 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 laser scanning microscope for scanning a laser beam onto a sample to detect fluorescence from the sample by a photodetector. 
     2. Description of the Related Art 
     In Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, a laser scanning microscope is disclosed including a first optical scanning system A for obtaining a scanned image of fluorescence from the sample and a second optical scanning system B for expressing peculiar phenomena such as cleavage of a caged reagent in a specific portion of the sample. 
       FIG. 1  is a diagram showing a constitution of a conventional laser scanning microscope. A sample  79  is irradiated with laser beams from the second optical scanning system B in synchronization with the scanning of the laser beams of the first optical scanning system A, and changes of the sample  79  with an elapse of time can be measured. The synchronization is carried out, when a control unit  81  controls a laser shutter  63 , optical scanning unit  64 , and photoelectric conversion device  70  of the first optical scanning system A, and a laser shutter  72  and optical scanning unit  73  of the second optical scanning system B. 
     The caged reagent and a fluorescent indicator having sensitivity to concentration of ions such as calcium ions are injected into the sample  79 . The sample  79  in which the caged reagent has been injected is irradiated with the laser beams from a laser unit  71  of the second optical scanning system B. A caged group of the caged reagent in the irradiated portion is cloven, and materials enclosed inside are released. The change of an ion concentration distribution in the sample  79  by this release is measured by a fluorescent image obtained by the laser beams from a laser unit  61  of the first optical scanning system A. With the cleavage of the caged reagent or by the irradiation with the laser beams of the second laser unit  71 , the fluorescent indicator of the sample  79  produces a certain degree of fluorescence. However, the control unit  81  controls an opening/closing timing of the laser shutters  63 ,  72  of each laser beam and a detection timing in the photoelectric conversion device  70  with the elapse of time. Therefore, a spectrum of fluorescence can be detected by a photodetector to obtain the fluorescent image without being influenced by the change of a fluorescent intensity from the fluorescent indicator with the cleavage of the caged reagent. 
     However, in the laser scanning microscope including first and second optical scanning systems described in the Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, there is possibility that the laser beams of the second optical scanning system are detected by the photodetector of the first optical scanning system. This has left much room for improvement in obtaining a desired fluorescent image. 
     For example, the use of a UV pulse laser (wavelength of 351 nm) as the laser unit  71  of the second optical scanning system B for cleaving the caged reagent is considered. Since much light intensity is required for cleaving the caged reagent, a reflected light of the laser beams of the second optical scanning system from the irradiated sample  79  is also intense. A dichroic mirror  75  does not sufficiently absorb the reflected light of the UV pulse laser beams, and a slight amount of the light is transmitted through an optical path of the first optical scanning system A. However, in a dichroic mirror  62  and filters such as a laser cut filter  67  usually for use in the first optical scanning system A, that is, an optical scanning system for acquiring images, transmission capabilities with respect to a short wavelength band of the UV laser are hardly considered. The wavelength of the UV pulse laser is reflected, transmitted, and detected by the photoelectric conversion device  70 , and a clear fluorescent image cannot be obtained. 
     Similarly, the use of an IR pulse laser (wavelength of 710 nm) as the laser unit  71  of the second optical scanning system B for cleaving the caged reagent is considered. It is to be noted that this IR pulse laser is assumed as laser capable of causing two photon excitation. Also for the IR pulse laser, the intense reflected light from the sample  79  is not sufficiently reflected by the dichroic mirror  75 , and the slight amount of the light passes through the optical path of the first optical scanning system A. For the filters usually for use in the first optical scanning system A, that is, the optical scanning system for acquiring the images, a long path filter which reflects a short wavelength and transmits a long wavelength is used in many cases. For these laser cut filters, transmission characteristics in the long wavelength band of IR are not considered. Therefore, the wavelength of IR pulse laser beams, which is longer than that of the fluorescence, passes through the laser cut filter, and is detected by the photodetector. Therefore, the clear fluorescent image cannot be obtained. 
     Moreover, to prevent the above-described phenomenon, as described in the Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, it is considered that the control unit  81 , for example, shifts a timing of laser irradiation to control the first and second optical scanning systems, and influences of the laser beams of the second optical scanning system B are avoided. However, in this case, since it is necessary to simultaneously control the optical scanning system and an optical detection system at a high speed, a complicate control is required for realizing this. Furthermore, in the technique described in the Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, the sample cannot be irradiated with two types of laser beams at the same time. Therefore, when the changes of the sample  79  with the elapse of time are measured, real time characteristics drop. 
     BRIEF SUMMARY OF THE INVENTION 
     According to a first aspect of the present invention, there is provided a laser scanning microscope comprising: a first optical scanning system which scans a first laser light having a spectrum in a visible range on a sample to excite fluorescence; a first dichroic mirror which separates the fluorescence from the sample from an optical path of the first laser light; a photodetector which detects the fluorescence separated by the first dichroic mirror; an emission filter which is disposed between the first dichroic mirror and photodetector to cut off the first laser light and to transmit desired fluorescence; a second optical scanning system which introduces a second laser light having the spectrum in an ultraviolet or infrared region into a specific portion on the sample; and a laser cut filter which is disposed between the first dichroic mirror and detector to limit transmission of the second laser light. 
     Moreover, according to another aspect of the present invention, there is provided a laser scanning microscope comprising: a first optical scanning system which scans a first laser light for observing a sample on the sample; a first light branch device which branches a light from the sample from an optical path of the first laser light; a photodetector which detects the light from the sample separated by the first light branch device; a second optical scanning system which irradiates a specific portion on the sample with a second laser light for stimulating or operating the sample; and a wavelength selection device which is disposed between the first light branch device and photodetector and which includes a first function of transmitting a desired observation light and a second function of limiting transmission of the second laser light. 
     Advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING 
       The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention, and together with the general description given above and the detailed description of the embodiments given below, serve to explain the principles of the invention. 
         FIG. 1  is a diagram showing a conventional laser scanning microscope; 
         FIG. 2  is a diagram showing a laser scanning microscope according to a first embodiment of the present invention; 
         FIGS. 3A ,  3 B are diagrams showing transmittance wavelength characteristics of a filter; 
         FIGS. 4A ,  4 B,  4 C are diagrams showing the transmittance wavelength characteristics of a dichroic mirror; 
         FIGS. 5A ,  5 B are concept diagrams showing characteristics and constitution of a laser cut filter; 
         FIG. 6  is a diagram of the laser scanning microscope according to another embodiment; and 
         FIG. 7  is a diagram of the laser scanning microscope according to another embodiment. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A first embodiment of the present invention will be described.  FIG. 2  is a diagram of a laser scanning microscope according to the present invention. 
     The laser scanning microscope includes a first optical scanning system A and second optical scanning system B. The first optical scanning system A is an optical system for observation, which scans the surface of a sample  29  with a laser light  11   a  outputted from a first laser unit  11 . The second optical scanning system B is an optical system for expressing a peculiar phenomenon in a specific portion of the sample. That is, the second optical scanning system B irradiates an arbitrary position of the sample  29  with a laser light  21   a  outputted from a second laser unit  21  to release a caged reagent. 
     The first optical scanning system A includes the first laser unit  11 , dichroic mirror  12 , first laser shutter  13 , first optical scanning unit  14 , pupil projection lens  15 , and mirror  16 . Furthermore, an optical detection system C is disposed in a branched optical path of the dichroic mirror  12  of the first optical scanning system A. The optical detection system C includes a laser cut filter  31 , dichroic mirror  17 , mirror  18 , fluorescence emission filters  19   a  and  19   b , confocal lenses  110   a  and  110   b , confocal apertures  111   a  and  111   b , and photoelectric conversion devices  112   a  and  112   b.    
     The laser cut filter  31  has characteristics for absorbing a reflected light of the laser light  21   a  of the second optical scanning system B from the sample  29 . A UV light is usually used to release the caged reagent. Therefore, the following laser unit is considered to be used. 
     (a) A UV pulse laser (wavelength of 351 nm) is used as the second laser unit  21 . 
     (b) An IR pulse laser (wavelength of 710 nm) is used as the second laser unit  21 . It is to be noted that the IR pulse laser is assumed as a laser capable of causing a two photon excitation phenomenon. 
     Therefore, the laser cut filter  31  has the characteristics to absorb the laser light as described above. Concretely, a filter having the characteristics shown in  FIG. 3  is used. 
       FIG. 3A  is a diagram showing filter characteristics to cut off the UV pulse laser (wavelength of 351 nm), and  FIG. 3B  is a diagram showing the filter characteristics to cut off the IR pulse laser (wavelength of 710 nm). 
     The second optical scanning system B includes the second laser unit  21  for releasing the caged reagent, and a second laser shutter  22 , second optical scanning unit  23 , pupil projection lens  24 , and dichroic mirror  25 . Optical axes of the first and second optical scanning systems A, B are synthesized by the dichroic mirror  25 , and introduced into an image forming lens  26  and objective lens  27 . Focal positions of the pupil projection lens  15  and pupil projection lens  24  are disposed to agree with the focal position of the image forming lens  26 . The sample  29  is laid on a stage  28 . 
     Here, a supposed combination of the first laser unit  11  and second laser unit  21 , and the characteristics of the dichroic mirror  25  which agree with conditions will be described hereinafter. 
     When a visible continuous laser (wavelength of 488 nm) is used in the first laser unit  11 , and the UV pulse laser (wavelength of 351 nm) is used in the second laser unit  21 , for transmittance wavelength characteristics of the dichroic mirror  25 , as shown in  FIG. 4A , the visible continuous laser (wavelength of 488 nm) and fluorescence (wavelength of 530 nm) are transmitted, and the UV pulse laser (wavelength of 351 nm) is reflected. 
     When the visible continuous laser (wavelength of 488 nm) is used in the first laser unit  11 , and the IR pulse laser (wavelength of 710 nm) is used in the second laser unit  21 , for the transmittance wavelength characteristics of the dichroic mirror  25 , as shown in  FIG. 4B , the dichroic mirror  25  transmits the visible continuous laser (wavelength of 488 nm) and fluorescence (wavelength of 530 nm) and reflects the IR pulse laser (wavelength of 710 nm). 
     When the IR pulse laser (wavelength of 850 nm) is used in the first laser unit  11 , and the IR pulse laser (wavelength of 710 nm) is used in the second laser unit  21 , for the transmittance wavelength characteristics of the dichroic mirror  25 , as shown in  FIG. 4C , the IR pulse laser (wavelength of 850 nm) and fluorescence having a wavelength of 530 nm are transmitted, and the IR pulse laser (wavelength of 710 nm) is reflected. 
     It is to be noted that the IR pulse laser for use herein is a laser capable of causing a two photon excitation phenomenon. 
     The first laser shutter  13 , second laser shutter  22 , first optical scanning unit  14 , second optical scanning unit  23 , and photoelectric conversion devices  112   a  and  112   b  are connected to a control unit  211 . The control unit  211  is connected to a CRT display  212 . As described later, the control unit  211  synchronizes the irradiation of the sample  29  with the laser light from the second optical scanning system B with the scanning of the first optical scanning system A. 
     Next, a function of the laser scanning microscope will be described. The laser light  11   a  from the first laser unit  11  passes, when the first laser shutter  13  controlled to open/close by the control unit  211  is in an opened state. Subsequently, the laser light  11   a  is guided into the first optical scanning unit  14 , and controlled by the control unit  211  to be scanned in an arbitrary direction. The laser light  11   a  is further converged onto a section  210  of the sample  29  via the pupil projection lens  15 , mirror  16 , dichroic mirror  25 , image forming lens  26 , and objective lens  27  to two-dimensionally scan inside the section  210  of the sample. 
     A fluorescent indicator (e.g., fluo-3, excitation wavelength of 488 m, fluorescent wavelength of 530 nm) excited by the wavelength of the first laser unit  11  is injected in the sample  29 . When the section  210  of the sample is scanned by the laser light, the fluorescent indicator is excited to generate the fluorescence. The fluorescence incident upon the objective lens  27  travels in an opposite direction in the same optical path as that of the laser light, and is guided into the objective lens  27 , image forming lens  26 , and dichroic mirror  12 . The dichroic mirror  12  includes characteristics to reflect the light which has a wavelength longer than that of the laser light  11   a  from the first laser unit  11 . Therefore, the fluorescence is accordingly reflected by the dichroic mirror  12 , and introduced into the optical detection system C. 
     When the sample  29  is multiple-dyed in the optical detection system C, the fluorescence transmitted through the laser cut filter  31  is split into the fluorescence having each wavelength by the dichroic mirror  17 . Among the split lights, the light having the specific wavelength passes through the fluorescence emission filters  19   a  and  19   b , and is focused by the confocal lenses  110   a  and  110   b . Moreover, only the light from the section  210  of the sample is incident upon the photoelectric conversion devices  112   a  and  112   b  by the confocal apertures  111   a  and  111   b  disposed in positions optically conjugated with the section  210  of the sample. 
     Output signals from the photoelectric conversion devices  112   a  and  112   b  are guided into the control unit  211 . The output signals are converted to digital signals in synchronization with scanning control, and displayed on a screen of the CRT display  212  in accordance with a scanned position. The displayed image indicates the fluorescent image which is a two-dimensional distribution of a fluorescent luminance in the section  210  of the sample, that is, the distribution in the section  210  having an ion concentration. 
     On the other hand, the laser light  21   a  from the second laser unit  21  passes, when the second laser shutter  22  controlled to open/close by the control unit  211  is in the opened state. The laser light  21   a  proceeds on the same optical axis as that of the laser light  11   a  from the first optical scanning system A via the second optical scanning unit  23 , pupil projection lens  24 , and dichroic mirror  25 . Moreover, the laser light  21   a  passes through the image forming lens  26  and objective lens  27  to irradiate the section  210  of the sample  29 . At this time, since the control unit  211  controls the second optical scanning unit  23 , an irradiation position in the section  210  can be selected independently of the scanned position of the first optical scanning system A. 
     The sample  29  in which the caged reagent has been injected is irradiated with the laser light  21   a  from the second laser unit  21 . Then, the caged group of the caged reagent of the irradiated portion is cloven, and substances enclosed inside are released. The change of the ion concentration distribution in the sample  29  by the release can be measured by the fluorescent image obtained by the first optical scanning system A. 
     At this time, the reflected light including the laser light  21   a  from the second laser unit  21  reflected on the sample  29  proceeds in the same optical path as that of the fluorescence generated on the section  210  of the sample  29 . Several % of the reflected light including the laser light  21   a  which has reached the dichroic mirror  25  passes through the dichroic mirror  25  and is introduced into the optical path of the first optical scanning system A. The reflected light including the second laser light guided into the first optical scanning system A passes through the mirror  16 , pupil projection lens  15 , and optical scanning unit  14 , and is reflected by the dichroic mirror  12 , and guided into the optical path of the optical detection system. 
     The reflected light including the laser light  21   a  reflected by the dichroic mirror  12  is absorbed by the laser cut filter  31  which is disposed on the optical path of the optical detection system C beforehand and which has characteristics to absorb the laser light  21   a . Therefore, only the fluorescence passes through the laser cut filter  31 , and is detected by the photoelectric conversion devices  112   a  and  112   b.    
     Here, transmission characteristics of the laser cut filter  31  for use in the present embodiment will be described. 
     With respect to the intensity of the laser light  21   a  from the second optical scanning system B as an excitation laser light with which the sample  29  is irradiated, the intensity of the fluorescence generated from the sample  29  is very weak. Therefore, even when the laser light  21   a  reflected by the sample  29  is reflected by the sample or an optical system midway and accordingly attenuated, the intensity becomes 1000 times or more that of the fluorescence generated from the sample  29  and directed toward the devices  112   a  and  112   b . Therefore, in order to clearly acquire the fluorescent image without being influenced by the reflected laser light  21   a , as laser cut filter characteristics for transmitting the fluorescence, the transmittance of the reflected laser light  21   a  needs to be at least 0.01% or less. 
     Additionally, in order to realize the characteristics, an interference filter using a multilayered film coating is used. For the interference filter, a large number of layers different in refractive index and film thickness are superimposed on one another, and the interference filter controls the transmittance by optical interference. However, for the interference filter, it is difficult to realize flat transmittance characteristics with respect to the wavelength band. Therefore, usually, targeted transmission wavelength and cut-off wavelength are set, and the filter is manufactured so as to obtain desired characteristics with the wavelength. As a filter which has actually heretofore been manufactured as the laser cut filter for selecting a fluorescent wavelength, it has been general to set the transmittance to 0.01% or less with respect to only an excitation wavelength corresponding to the fluorescence. 
     That is, the conventional laser cut filter is a filter having “a function of extracting the fluorescence”, and is assumed not to include a function of cutting off the “second laser light” in a case where the second optical scanning system is disposed. The laser cut filter  31  for use in the present embodiment further includes this function. 
       FIG. 5A  is an explanatory view of the characteristics of the laser cut filter according to the present embodiment. 
     In  FIG. 5A , the first laser light is represented as the visible continuous laser (wavelength of 488 nm), the second laser light is represented as the UV pulse laser (wavelength of 351 nm), and the light from the sample is represented as the fluorescence from the sample (wavelength of 530 nm). 
     An upper part of  FIG. 5A  shows an intensity distribution of the first laser light, second laser light, and the light from the sample. As shown in this figure, the intensity of the second laser light is higher than that of the first laser light in many cases. This is because the second laser light is used for a purpose of exciting or operating the sample. It is seen from this that necessity of securely cutting off the second laser light rather than the first laser light is high. 
     A lower part of  FIG. 5A  shows a concept of the transmission characteristics of the laser cut filter according to the present embodiment. 
     As described above, the laser cut filter according to the present embodiment includes two functions. That is, the filter includes the transmission characteristics for realizing a first function which is a “function of extracting the fluorescence” and a second function which is a “function of cutting off the second laser light”. With thee two functions, it is possible to obtain a clear fluorescent image. 
     In order to realize the laser cut filter of the present embodiment, as shown in  FIG. 2 , the fluorescence emission filters  19   a  and  19   b  including the “function of extracting the fluorescence” (first function) and the laser cut filter  31  including the “function of cutting off the second laser light” (second function) are disposed. Moreover, as shown in  FIG. 5B , filter films  31   a ,  32   a  including the respective functions may be formed on opposite surfaces of one glass. The filters shown in  FIG. 5B  may be used instead of the filters  19   a  and  19   b  of  FIG. 2 . At this time, the filter  31  is not required. 
     When the laser cut filter  31  including the above-described transmission characteristics is used and incorporated in the optical detection system C in this manner, the laser light  21   a  included in the reflected light from the sample  29  can securely be removed, and the clear fluorescent image is obtained. The simultaneous irradiation with the first and second laser lights  11   a  and  21   a  is also possible. Furthermore, when the sample  29  is multiple-dyed, as shown in  FIG. 2 , the laser cut filter  31  may be disposed in a common optical path of the optical detection system C, and therefore the system can easily be constituted. 
     A second embodiment of the present invention will be described.  FIG. 6  is a diagram of the laser scanning microscope according to the present invention. The same components as those of the first embodiment are denoted with the same numerals, and detailed description thereof is omitted. 
     In the second embodiment, a UV pulse laser  34  and an IR pulse laser  35  whose wavelength can be varied and which can cause the two photon excitation phenomenon are used in the laser of the second optical scanning system B. Moreover, these lasers  34 ,  35  can be selected and used by controlling the opening/closing of laser shutters  36  and  37 . The dichroic mirror  25  is disposed in a position where the optical axis of the laser light  11   a  from the first optical scanning system A and that of laser light  34   a  or  35   a  from the second optical scanning system B are synthesized. At least one dichroic mirror  25  is disposed in an electromotive turret  32  in which a plurality of dichroic mirrors can be disposed. 
     Further to cut off the laser light  34   a  or  35   a  from the laser unit of the second optical scanning system B, the laser cut filter  31  is disposed on the optical path of the optical detection system C. At least one laser cut filter  31  is disposed in an electromotive turret  33  in which a plurality of filters can be disposed. 
     It is to be noted that the electromotive turrets  32  and  33  are usually of a rotary type, but may be of a slider type if necessary. 
     Moreover, the electromotive turrets  32  and  33  and laser shutters  36 ,  37  are connected to the control unit  211 , and can be controlled by the control unit  211 . 
     The function of the laser scanning microscope constituted in this manner will be described. The UV pulse laser  34  is used as the laser unit of the second optical scanning system B. The laser light  11   a  from the first laser unit  11  of the first optical scanning system A, and the UV pulse laser light  34   a  outputted from the laser unit of the second optical scanning system B pass through the respective optical devices in the same manner as in the first embodiment. Moreover, the optical axes of the laser light  11   a  from the first optical scanning system A and the UV pulse laser light  34   a  from the second optical scanning system B are synthesized by the dichroic mirror  25 . The dichroic mirror  25  includes characteristics to transmit the laser light  11   a  from the first optical scanning system A and to reflect the UV pulse laser light  34   a  which is the laser light from the second optical scanning system B. The dichroic mirror  25  is disposed on the optical path by the electromotive turret  32  which operates in conjunction with the opening/closing operation of the laser shutter  36 . 
     The respective laser lights from the first and second laser units synthesized by the dichroic mirror  25  pass through the image forming lens  26  and objective lens  27 , and are focused on the section  210  of the sample  29  in the same manner as in the first embodiment. The caged reagent is released by the UV pulse laser light  34   a , and the fluorescent indicator is excited by the laser light  11   a  from the first optical scanning system A to generate the fluorescence. 
     The fluorescence generated from the sample  29  and the UV pulse laser light  34   a  which is the reflected light from the sample travel in an opposite direction through the optical path of the first optical scanning system A, and the fluorescence and the reflected light of the UV pulse laser light  34   a  are introduced into the optical detection system C via the dichroic mirror  12 . 
     Among the fluorescence and UV pulse laser light  34   a  introduced into the optical detection system C, the fluorescence passes through the laser cut filter  31 , and the UV pulse laser light is absorbed by the laser cut filter  31 . It is to be noted that the electromotive turret  33  operates in conjunction with the opening/closing operation of the laser shutter  36 , and the laser cut filter  31  including the transmission characteristics to absorb the UV pulse laser light is disposed beforehand on the optical path. 
     The fluorescence which has passed through the laser cut filter  31  passes through the respective optical devices in the same manner as in the first embodiment. Moreover, the fluorescence is detected by the photoelectric conversion devices  112   a  and  112   b . The detection signal is processed by the control unit  211 , and subsequently displayed on the CRT display  212 . 
     On the other hand, in order to release the caged reagent or to light-discolor the sample in which protein (e.g., YFP) is expressed, the IR pulse laser  35  is sometimes used as the laser light of the second optical scanning system B. In this case, the dichroic mirror  25  for synthesizing the optical axes of the laser light from the first optical scanning system A and the IR pulse laser light  35   a  from the second optical scanning system B includes the transmission characteristics to transmit the laser light  11   a  from the first optical scanning system A and reflect the IR pulse laser light  35   a . Moreover, in conjunction with the opening/closing operation of the laser shutter  37 , the electromotive turret  32  brings the dichroic mirror  25  onto the optical path beforehand. 
     Moreover, also in the optical detection system C, the laser cut filter  31  includes the transmission characteristics to transmit the fluorescence and absorb the IR pulse laser light  35   a  which is the reflected light. Furthermore, the electromotive turret  33  brings the laser cut filter  31  onto the optical path beforehand in conjunction with the opening/closing operation of the laser shutter  37 . 
     In this manner, in the second embodiment, the dichroic mirror  25  for synthesizing the optical axes of the laser lights from the first and second optical scanning systems A and B is disposed on the electromotive turret. Furthermore, in the optical detection system C, the laser cut filter  31  for absorbing the laser light of the second optical scanning system B which is the reflected light from the sample  29  is disposed in the electromotive turret  33 . Moreover, the electromotive turrets  32  and  33  are operated in conjunction with the opening/closing operation of the laser shutters  36  and  37 . Accordingly, there can be provided a system in which either the UV pulse laser  34  or IR pulse laser  35  can be selected and used as the laser unit of the second optical scanning system B for use in releasing the caged reagent. 
     Moreover, when a manual turret is used instead of the electromotive turrets  32  and  33 , a system including the similar function can inexpensively be provided. 
     A third embodiment of the present invention will be described.  FIG. 7  is a diagram of the laser scanning microscope according to the present invention. The same components as those of the first and second embodiments are denoted with the same reference numerals and the detailed description is omitted. 
     The laser scanning microscope of the present embodiment includes a constitution in which an image forming lens  41  of the first optical scanning system A and an image forming lens  43  of the second optical scanning system B for observation are independently disposed, and the objective lens  27  is shared. 
     The first optical scanning system A is constituted as a laser scanning microscope D. The first optical scanning system A is constituted of the first laser unit  11 , the first laser shutter  13 , the dichroic mirror  12 , the first optical scanning unit  14 , the pupil projection lens  15 , the image forming lens  41 , a dichroic mirror  42 , and the objective lens  27 . At least one dichroic mirror  42  is disposed in an electromotive turret  47  in which a plurality of dichroic mirrors can be disposed. Furthermore, the optical detection system C is disposed on the branched optical path of the dichroic mirror  12  of the first optical scanning system A. Since the optical detection system C is the same as that of the second embodiment, the description is omitted. 
     The dichroic mirror  42  disposed in the electromotive turret  47  of the first optical scanning system A includes characteristics to reflect the wavelength of the laser light from the first optical scanning system A and the light having the long wavelength and to transmit the laser light from the second optical scanning system B. 
     The second optical scanning system B is constituted as an illuminative light introduction apparatus E. The second optical scanning system B is constituted of the second laser unit  21 , second laser shutter  22 , second optical scanning unit  23 , pupil projection lens  24 , and image forming lens  43 , and a mirror  44 . 
     It is to be noted that the second optical scanning unit  23  may be omitted to constitute the second optical scanning system B. 
     The second laser shutter  22  and second optical scanning unit  23  are controlled by a second control unit  45 . The second control unit  45  is connected to a first control unit  46  for controlling synchronization with the first optical scanning system A. 
     It is to be noted that the second control unit  45  is not necessarily required. The second laser shutter  22  and second optical scanning unit  23  may also directly be connected to the first control unit  46 . 
     Moreover, in the present embodiment, the laser scanning microscope D and illuminative light introduction apparatus E are constituted as independent units, and are structured to be attachable/detachable, for example, by a dovetail structure or bolt fastening. 
     Next, the function of the laser scanning microscope constituted in this manner will be described. The laser light  11   a  emitted from the first laser unit  11  of the first optical scanning system A passes through the respective optical devices of the first optical scanning system A, and is formed into a parallel light by the image forming lens  41 . Moreover, the laser light  11   a  is reflected by the dichroic mirror  42  and focused by the objective lens  27  to scan on the section  210  of the sample  29 . The fluorescence from the section  210  of the sample  29  travels forward in an optical path similar to that described in the first embodiment, and is detected by the optical detection system C. 
     On the other hand, the laser light  21   a  emitted from the laser unit  21  of the second optical scanning system B passes through the respective optical devices of the second optical scanning system B, and is formed into the parallel light by the image forming lens  43 . Moreover, the laser light  21   a  is reflected by the mirror  44  and synthesized with the optical axis from the first optical scanning system A via the dichroic mirror  42 . Furthermore, the laser light  21   a  is focused by the objective lens  27  to irradiate the section  210  of the sample  29 . 
     When the first control unit  46  controls the second control unit  45  and second optical scanning unit  23 , an irradiation position and range by the second optical scanning system B can be selected independently of a scanning position and range of the first optical scanning unit  14 . 
     As described above, in the third embodiment, the first optical scanning system A includes the image forming lens  41 , and the second optical scanning system B includes the image forming lens  43 . Therefore, the laser light which has passed through the image forming lenses  41 ,  43  forms the parallel light, and the optical axis of the first optical scanning system A can easily be matched with that of the second optical scanning system B. 
     That is, a luminous flux of a connecting portion of the laser scanning microscope D including the first optical scanning system A with respect to the illuminative light introduction apparatus E including the second optical scanning system B is the parallel light. Therefore, optical axis alignment is facilitated in connecting the laser scanning microscope D to the illuminative light introduction apparatus E. 
     Moreover, the first optical scanning system A is constituted as the laser scanning microscope D, and the second optical scanning system B can be constituted as the illuminative light introduction apparatus E. Therefore, since the respective apparatuses can be constituted as different appropriates, the illuminative light introduction apparatus E can be provided as an apparatus for upgrading the system of the laser scanning microscope D. 
     Furthermore, when the illuminative light introduction apparatus E includes the constitution without including the optical scanning unit  23  of the second optical scanning system B, the apparatus can be provided as an inexpensive apparatus which is easily controlled. 
     Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general invention concept as defined by the appended claims and their equivalents.

Technology Classification (CPC): 6