Patent Publication Number: US-8531762-B2

Title: Confocal microscope apparatus

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application is a Divisional Application of U.S. application Ser. No. 12/689,523, filed Jan. 19, 2010, now U.S. Pat. No. 8,072,680, which is a Divisional Application of U.S. application Ser. No. 11/677,444, filed Feb. 21, 2007, now U.S. Pat. No. 7,675,676, which is a Divisional Application of U.S. application Ser. No. 10/393,721, filed Mar. 21, 2003, now U.S. Pat. No. 7,196,843, which is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2002-89878, filed Mar. 27, 2002, the entire contents of all of which are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to a confocal microscope apparatus which excites a specimen which has been marked with a fluorescent dye or fluorescent protein using the excitation wavelength, and detects fluorescence emitted from the specimen. 
     2. Description of the Related Art 
     A scanning laser microscope has been proposed, which includes a first optical scanning system for obtaining a scan image of a sample and a second optical scanning system for causing a particular phenomenon in specific areas on the sample surface (refer to Jpn. Pat. Appin. KOKAI Publication No. 2000-275529, the entire contents of which are incorporated herein by reference). In this laser scanning microscope, a specific area on the sample surface is irradiated using a laser light source and an optical path of the first optical scanning system, thus stimulating the sample or a chemical substance injected into the sample. A specific area on the sample surface which is different from the above-mentioned area is excited using a laser light source and an optical path of the second optical scanning system, and the fluorescence is detected, and imaging is carried out. In the specification, unless stated otherwise, an optical scanning system for obtaining images of a sample is called a “first optical scanning system” and an optical scanning system for causing a particular phenomenon in specific areas of a sample is called a “second optical scanning system”. 
     Generally, in the confocal microscope, the focal point on the sample surface and the conjugated focal point thereof are provided before the detection device, and a pinhole is provided therein. Thereby, the resolution of the sample along the depth direction is 1.22 λ/NA, and a smaller confocal effect is being utilized than when a regular microscope is used for observation. There is resolution as a result of this confocal effect, and thus a sharp cross sectional image (that is, an image to obtain a thin slice image along depth direction) can be obtained for the sample which is being scanned. 
     When the image is taken at a high speed or when a dark sample is being used, the confocal effect is weakened by opening the pinhole (that is, enlarging a diameter of the pinhole), and the image is made bright by lowering the resolution of the fluorescence. 
     Thus the confocal microscope has the pinhole and decreases the resolution, and thus depth-direction information can be obtained. However, since the focal depth of the sample is determined by the flux diameter of the coherent light which is irradiated on the objective lens, it is impossible to change the focal depth at the pinhole. 
     Meanwhile, Koehler illumination is often used as the lighting to the sample by the microscope. This Koehler illumination along the thickness direction of the cross section of the sample causes almost uniform excitation. 
     In the conventional confocal microscope described above, when the apparatus is realized by using 2 laser scanning paths and one objective lens, the excitation light intensity distribution along the depth direction on the sample surface of the laser beam for sample stimulation and the laser beam for obtaining images are almost the same since only wavelength differences are generated. 
     BRIEF SUMMARY OF THE INVENTION 
     A confocal microscope apparatus according to a first aspect of the present invention is characterized by comprising: a first optical scanning system which obtains a scan image of a sample using a laser beam from a first laser light source; a second optical scanning system which scans specific regions of a sample with a laser beam from a second laser light source that is different from the first laser light source, thereby causing a particular phenomenon; and a beam diameter varying mechanism which can change the beam diameter of the laser beam of at least one of the first optical scanning system and the second optical scanning system. 
     A confocal microscope apparatus according to a second aspect of the present invention is characterized by comprising: a first optical scanning system which scans a sample via an objective lens with incoherent light output from an incoherent light source, and detects fluorescence emitted from the sample via the objective lens; and a second optical scanning system which irradiates specific regions of the sample with laser beam output from a laser light source, thereby causing a particular phenomenon, in which the first optical scanning system further comprises a rotatable disk to obtain a confocal effect, the light output from the incoherent source scans the sample via the rotatable disk, and the fluorescence is detected via the rotatable disk. 
     A confocal microscope apparatus according to a third aspect of the present invention is characterized by comprising: a first optical system which illuminates a sample via an objective lens with incoherent light output from an incoherent light source, and detects fluorescence emitted from the sample via the objective lens; and a second optical scanning system which irradiates specific regions of a sample with a laser beam from a laser light source, thereby causing a particular phenomenon. 
     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 schematic diagram of a confocal microscope apparatus according to a first embodiment of the invention; 
         FIG. 2  is a view showing a structural example of a first beam diameter varying mechanism and a second bean diameter varying mechanism; 
         FIG. 3  is a schematic diagram of a confocal microscope apparatus according to a second embodiment of the invention; 
         FIG. 4  is a view showing an example of a rotatable disk used in the invention; and 
         FIG. 5  is a view schematically showing a nerve tissue observation. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Embodiments of the present invention will be described with reference to the drawings. 
     First Embodiment 
       FIG. 1  is a schematic diagram of a confocal microscope apparatus according to a first embodiment of the invention. 
     In  FIG. 1 , the confocal microscope apparatus comprises: a first optical scanning system  100  for observation (or for obtaining images) which scans a focal surface of a sample  134  with a laser beam from a first laser light source  101 ; and a second optical scanning system  200  for radiating the laser beam output from the second laser light source  201  onto an optional position on the sample  134 , and splitting the caged reagent (i.e. for sample stimulation). An optical path of the first optical scanning system  100  and an optical path of the second optical scanning system  200  meet at a dichroic mirror  120 . As a result, the first optical scanning system  100  and the second optical scanning system  200  share an objective lens  132 . 
     In the first optical scanning system  100  and the second optical scanning system  200 , the coherent light output from the first laser light source  101  arrives at the dichroic mirror  120  by way of a first beam diameter varying mechanism  102  and a first optical scanning unit  104 . Also, the coherent light output from the second laser light source  201  reaches the dichroic mirror  120  by way of a beam diameter varying mechanism  202  and a second optical scanning unit  203 . 
     In addition, the first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202  are connected electrically or indirectly to an excitation light intensity calculator  160 . As a result, the excitation light intensity calculator  160  can obtain beam diameter information of the beams output from the first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202 . 
     The first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202  may include, as shown in  FIG. 2 , for example, a plurality of mechanisms which change the flux diameter, such as beam expanders, on a rotatable turret. Also, mechanisms in which optical elements such as a plurality of lenses are combined and the flux diameter is changed while the coherence of the laser is maintained (for example, a zoom mechanism) may be adopted as the first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202 . 
     The operation of the confocal microscope apparatus according to the first embodiment, which has the above-described configuration, will be described. 
     The first optical scanning system  100  and the second optical scanning system  200  are used for radiating a coherent light at an optional (desired) position on the sample  134 . Specifically, this is as described below. 
     That is, the flux diameter of the coherent light generated from the first laser light source  101  and the second laser light source  201  respectively, are varied (adjusted) with the first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202 . 
     The light beam output from the first beam diameter varying mechanism  102  passes a dichroic mirror  150 , and is arbitrarily deflected to an XY direction by each of scanning mirrors  104   a  and  104   b  of the first optical scanning unit  104 . The deflected light beam is reflected at the mirror  106  after passing through a relay lens  105 , and then irradiated onto the dichroic mirror  120 . Meanwhile, the light beam output from the second beam diameter varying mechanism  202  is suitably deflected in an XY direction by each of scanning mirrors  203   a  and  203   b  of the second optical scanning unit  203 . The deflected light beam passes through the relay lens and is irradiated onto the dichroic mirror  120 , and the optical path is deflected at the dichroic mirror  120 . 
     In addition, the coherent light from the dichroic mirror  120  is irradiated onto an image formation lens  130 . By changing the flux diameters of the laser beams from the first laser light source and the second laser light source at the first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202  with respect to the pupil diameter of the objective lens  132 , the width of the excitation light distribution (and/or the intensity distribution) along the depth direction on the surface of the sample  134  corresponding to each of the optical scanning systems can be changed. 
     The light beam that has passed through the image formation lens  130  reaches the objective lens  132 , passes through the objective lens  132  and is focused on an arbitrary cross section  138  of the sample  134  which is mounted on a stage  136 . The stage  136  is movable along the XY horizontal direction and the height direction (Z axis direction—the direction of the arrow in  FIG. 1 ). 
     As described above, when the sample  134  is being scanned, in accordance with the application, a particular field may be scanned by each of the scanning mirrors  203   a  and  203   b  or it may be kept still and irradiated in spots. Further by skipping each of the scanning mirrors  203   a  and  203   b  momentarily, the field can be irradiated in spots at a number of arbitrary positions from moment to moment. Meanwhile, the coherent light generated from the first laser light source  101  is transmitted by the dichroic mirror  150  as described above, and it is deflected by each of the scanning mirrors  104   a  and  104   b  of the first optical scanning unit  104 . 
     When light beam is irradiated on the sample  134  by the first optical scanning system  100 , the fluorescent marker chemical is excited and fluorescence is generated. 
     The fluorescence from the sample  134  takes the opposite direction of the optical path of the light irradiated on the sample  134  and passes from the objective lens  132  by way of the image formation lens  130 , the dichroic mirror  120 , the first optical scanning unit  104 , the relay lens  103 , each of the scanning mirrors  104   a  and  104   b , and arrives at the dichroic mirror  150 . The fluorescence is reflected and incident to a photometry filter  140 . 
     The light beam is incident to the photometry filter  140  and only the fluorescent wavelength from the sample  134  is selected, and the light beam from the sample  134  having only the fluorescence wavelength is focused at a surface of the pinhole  144  by a lens  142 . The fluorescence, which has passed through the pinhole  144 , is measured by a photoelectric conversion device  146 . 
     The excitation light intensity calculator  160  calculates the excitation light intensity distribution on the sample surface by inputting the information on the beam diameter of the beam output by the first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202  and the performance (specification) of the objective lens being used at the time. It also has other functions such as outputting values, which have already been stored in a memory, to interfaces such as a computer or a display (not shown in the figure). 
     According to the confocal microscope apparatus of the first embodiment of the invention as mentioned above, when the sample  134  is observed and recorded by the first optical scanning system  100 , by irradiating coherent light on the sample  134  by the second optical scanning system  200 , the dynamics (chemical reactions) of sample  134  which are caused by the coherent light irradiation by the second optical scanning system  200  can be adjusted by the first optical scanning system  100 . 
     In this case, in the first embodiment, the excitation light distribution along the depth direction on the sample surface by the first optical scanning system  100  and the second optical scanning system  200  are independently set by the first beam diameter varying mechanism  102  and the second beam diameter varying mechanism  202 . Accordingly, even if the excitation light distribution is narrow for the field of the sample being excited by the second optical scanning system, that is, even in the case where a large area of the sample along the thickness direction is excited, by broadening the excitation light distribution of the first optical scanning system, it is possible to carry out observation. 
     Also, unlike the case described above, at the second optical scanning system, a wide field of the sample along the thickness direction is stimulated, and at the first optical scanning system, the excitation light distribution field along the thickness direction is narrowed, and thus the cross section  138  of the sample can be observed with high resolution. 
     The first embodiment may be configured such that an IR pulse laser is used as the first laser light source  101 , and a fluorescent image is obtained by two photon absorption. In this case, the two photon absorption phenomenon occurs only at the position where the image is formed and theoretically the pinhole  44  is unnecessary. Also, because the dichroic mirror  150  can transmit the IR pulse laser, reflect the visible fluorescence and lead it to the photoelectric converter  146 , this embodiment has the property of reflecting short wavelengths. It is also configured such that the beam diameter varying mechanism  102  is not used. 
     As described above, by using an IR pulse laser as the first laser light source  101 , the configuration of the first optical scanning system  100  is simplified. In addition, even in the case where the first beam diameter varying mechanism  102  is not used, the width of the excitation light distribution along the depth direction on the sample surface of the optical scanning system  1  becomes narrow than the excitation light distribution along the depth direction of the second optical scanning system  200  due to the two photon absorption phenomenon. Further, in the case where the thickness of the sample to be stimulated is to be changed, the width of the excitation light distribution of the second optical scanning system  200  can be made smaller by the second beam diameter varying mechanism  202 . 
     Second Embodiment 
     A confocal microscope according to a second embodiment of the invention is described with reference to  FIG. 3 .  FIG. 3  is a schematic diagram of the confocal microscope apparatus according to the second embodiment of the invention. The second optical scanning system  200  of  FIG. 3  is the same as that of the first embodiment, and has been assigned the same reference numbers and thus detailed descriptions thereof are omitted. 
     In the second embodiment, a first optical scanning system  100 ′ has an incoherent light source such as a mercury light source, a halogen light source, or an LED light source as a light source  301 . An optical lens  302 , a polarizing plate  303  and a polarizing beam splitter (PBS)  304  are arranged on an optical path of a light beam emitted from the light source  301 . 
     A rotatable disk  305 , a first image formation lens  307 , a quarter wave plate  308 , and an objective lens  309  are arranged on a reflection optical path of the PBS  304 , and a light beam from the light source is incident to a sample  310  by way of these elements. 
     The rotatable disk  305  is connected to a shaft of a motor (not shown) via a rotation shaft  306 , and rotates at a predetermined rotation speed. The rotatable disk  305  has linear transmit portions through which light passes, and linear shield portions which shield light, and the linear transmit portions and the linear shield portions are alternately arranged. In addition, the line width of the shield portion is wider than that of the transmit portion, and for example, the ratio of the width of the shield portion to that of the transmit portion is 1:9 (refer to  FIG. 4 ). 
     If the width of the portion through which light passes is W, and as is the case with the pinhole, assuming that magnification with which the specimen image is projected onto the disk is M, the wavelength of the light is λ, and the numerical aperture of the objective lens is NA,
 
 W=kλM/NA  
 
where k is a coefficient and a value in the range of 0.5 to 1 is often used for k.
 
     Also, a CCD camera  312  is arranged on the transmission optical path of the PBS  304  via a second image formation lens  311 . A monitor  313  for observing the image taken by the CCD camera  312  is connected to the camera  312 . 
     The operation of the confocal microscope of the second embodiment having the above configuration will be described in the following. 
     The light beam output from the light source  301  passes through the optical lens  302 , and at the polarizing plate  303  it is transformed to linearly polarized light having only a predetermined polarization, and then input into the PBS  304 . The PBS  304  reflects the deflected light beam in the direction in which the beam has passed through the polarizing plate and a light in a direction parallel thereto is transmitted. 
     The light beam reflected at the PBS  304  is input into the rotatable disk  305  which rotates at a predetermined speed. The light beam passing through the transmit portion of the rotatable disk  305  passes through the first image formation lens  307  and becomes circularly polarized at the quarter wave plate  308 , and is focused with the objective lens  309  to be irradiated on the sample  310 . 
     The light beam reflected by the sample  310  passes through the objective lens  309 , and at the quarter plate  308  it becomes linearly polarized light which is orthogonal to that at the time of input, thereby focusing the image of the sample  310  on the rotatable disk  305 , via the second imaging lens  311 . 
     The focused component of the formed image formed on the rotatable disk  305  passes through the transmit portion of the rotatable disk  305 . The component passing through the rotatable disk  305  is transmitted by the PBS  304 , and arrives at the CCD camera  312  by way of the second image formation lens  311 . The specimen image is formed on the image formation surface (image pickup surface). 
     If a particular moment when the sample  310  is being observed is considered, a line is projected on the sample  310  along a particular direction as shown in  FIG. 4 . 
     In this situation, in the case where the light beam reflected from the sample  310  in this state is focused on the rotatable disk  305 , a line is projected on the rotatable disk  305  for the portion of the sample  310  which is in focus. However, for the unfocused portion, the image that is projected on the rotatable disk  305  is blurred, and thus most of the unfocused image cannot be transmitted. Accordingly, only images which are in focus are transmitted to the rotatable disk  305 . 
     When the rotatable disk  305  is not rotating, the situation is not changed and the image is simply one in which the sample and the line overlap. However, by rotating the rotatable disk  305 , the line which includes the transmit portion and the shield portion moves while changing its direction on the sample  310 , and thus there is uniformity, the line image disappears and an image which is in focus can be observed. Thus, if the rotation of the rotatable disk  305  is sufficiently fast with respect to the exposure time of the CCD camera  312 , the focused image can be picked up by the CCD camera  312  and observed at the monitor  313 . 
     For example, if the CCD camera  312  has a TV rate as a usual, the exposure time is 1/60 second or 1/30 second. Therefore, the number of rotations of the rotatable disk  305  during the exposure time should be about 1800 rpm at which half revolutions occur. 
     The excitation light distribution along the depth direction on the surface of the sample  310  of the first optical scanning system  100 ′ at this time is the same as the light distribution of Koehler illumination of the microscope in the longitudinal direction of the slit. At the width direction of the slit, the distribution is the same as the second optical scanning system. 
     Accordingly, excitation light distribution along the depth direction on the sample surface of the first optical scanning system is a distribution of which both longitudinal direction and width direction distributions are combined. It is possible to change the excitation light intensity distribution along the depth direction, by varying the width of the slit and the space between the slits of the rotatable disk  305 . 
     In the second embodiment, by detecting the dynamic change which caused reaction of the light radiated by the second optical scanning system which has been shown in the first embodiment using the first optical scanning system  100 ′, the excitation light distribution along the depth direction on the surface of the first and second samples can be different. Accordingly, a wider field of measurement is possible in the first optical scanning system  100 ′ than the stimulation field in the second optical scanning system  200 . 
     Particularly in nervous system measurements, in order to catch movements of the nerve which extend along the thickness direction of the sample, it is necessary to obtain the images with high speed. Usually, with the confocal microscope apparatus, in order to increase the width of the excitation light distribution along the depth direction on the surface of the sample, if the sample is extends along the thickness direction, the image cannot be captured with one measurement. As a result, as in the second embodiment, by reducing the width of the excitation light distribution along the depth direction on the surface of the sample, image measurements for wider fields can be taken. Accordingly, the second embodiment may have a configuration in which the rotatable disk  305  is omitted. Also the rotatable disk is not limited to the structure shown in  FIG. 4 . Provided that the confocal effect can be obtained, any configuration or structure can be used. For example, the rotatable disk may be one having pinholes formed therein, and it can be a reflection type rotatable disk rather than the transmit type of the above-described embodiment. 
     In addition, in the second embodiment, the second beam diameter varying mechanism  202  is not necessarily needed. However, if the second embodiment has the second beam diameter varying mechanism  202 , it is possible to change the proportion of the first cross section and the second cross section, and by fine adjustment of the field for obtaining images and the portion for stimulation, the degree of freedom of the experiment (and/or observation) is increased. In addition, when the second beam diameter varying mechanism  202  is provided, it is preferable that the excitation light intensity distribution calculator  160  is provided as in the case of the first embodiment. 
     Also, in the above-described configuration, by the first optical scanning system  100 ′ having an optical microscope system with Koehler illumination, it becomes possible for the image to be obtained in a wider excitation field. In this case, the rotatable disk  305  is unnecessary. 
     In the above-described second embodiment, the PBS  304  may be replaced with a dichroic mirror. In this case, the light beam from the light source is reflected at the dichroic mirror, and the fluorescence from the sample passes through the dichroic mirror. Thus the optical path of the optical excitation system and that of the optical measurement system can be separated, and as a result the polarizing plate  303  is unnecessary. 
     Applications of the confocal microscope apparatus of each of the above-described embodiments include for example, the application in the field of cell research in which the cell is locally excited and reactions at the excited regions are observed. 
     In the method known as the uncaged method, by locally exciting the cell, the concentration of the activated material is changed. When this change in concentration is to be measured, by measuring peripheral portions other than the locally excited regions simultaneously, the internal functions of the cell can be analyzed. 
     In the photo-bleach method, by locally exciting the cell, the excited regions are discolored. The phenomenon is seen where due to migration of proteins from the periphery, over time color returns to the region which has been discolored. Accordingly, measurements for both the locally stimulated region and the peripheral portions are necessary. 
     An example thereof is shown using  FIG. 5 .  FIG. 5  is a schematic view showing a nerve tissue observation. 
     For example, when ions transmitted on an axis cylinder  3  from a cell body  1  to a cell body  2  are observed with the caged fluorescent dyes introduced into the cell body  1  as a probe, first a laser beam for stimulating a sample is radiated on a focal point surface  4  of the cell body  1 . Next, subsequent changes are observed with a laser beam for sample observation. However, the excitation light intensity distribution of the laser beam for sample observation along the depth direction usually has the same depth as the excitation light intensity distribution  5  of the laser beam for sample stimulation. Thus, in the prior art, the fluorescent dye which transmits the axis cylinder  3  and is not within that distribution field cannot be observed because it is not exposed to excitation light. To the contrary, in each of the embodiments of the invention, the excitation light intensity distribution along the depth direction, of the laser beam for sample stimulation and the laser beam for obtaining images on the surface of the sample are each independently varied, thus solving the problem of the prior art. 
     The inventions described in the following are extracted from the embodiments described below. The above-described embodiments do not limit the invention. Accordingly various modifications may be made within the scope of the general inventive concept of the invention. 
     The confocal microscope apparatus according to a first aspect of the present invention is characterized by comprising: a first optical scanning system which obtains a scan image of a sample using a laser beam from a first laser light source; a second optical scanning system which scans specific regions of a sample with a laser beam from a second laser light source that is different from the first laser light source, thereby causing a particular phenomenon; and a beam diameter varying mechanism which can change the beam diameter of the laser beam of at least one of the first optical scanning system and the second optical scanning system. By combining the optical laser system and the laser scanning microscope, it becomes possible to change the width of measurement by using differences in the excitation intensity distribution along the depth direction on the surface of the sample. Specifically, this is done in the following manner. 
     Conventionally, when movement of the sample is being analyzed, it is of course desirable for the field of excitation and the field for obtaining the images to be different, and also for the excitation light intensity distribution on the sample surface of the laser beam for sample stimulation along the depth direction and the excitation light intensity distribution on the sample surface of the laser beam for obtaining images along the depth direction to be different from each other. In addition, it is desirable for the width of the excitation light intensity distribution along the depth direction to be intentionally made small. 
     In the first aspect, a beam diameter varying mechanism for changing the beam diameter of the laser beam is provided to the output exit for the laser beam of each of the optical scanning systems. When the flux diameter is reduced by this beam diameter varying mechanism, the numerical aperture of the objective lens is smaller than in the case where the flux diameter is large. Consequently, the width of the excitation light intensity distribution along the depth direction on the surface of the sample can be reduced without changing the objective lens. Further, by providing each of the optical systems with the beam diameter varying mechanism, the excitation light intensity distribution along the depth direction of the sample surface of each of the optical systems can be changed independently. Also, the excitation light distribution along the depth direction on the sample surface can be changed intentionally. 
     The confocal microscope apparatus according to a second aspect of the present invention is characterized by comprising: a first optical scanning system which scans a sample via an objective lens with incoherent light output from an incoherent light source, and detects fluorescence emitted from the sample via the objective lens; and a second optical scanning system which irradiates specific regions of the sample with laser beam output from a laser light source, thereby causing a particular phenomenon, in which the first optical scanning system further comprises a rotatable disk to obtain a confocal effect, the light output from the incoherent source scans the sample via the rotatable disk, and the fluorescence is detected via the rotatable disk. The optical laser system and the disk type confocal microscope apparatus are combined, so that it becomes possible to change the width for measurement due to differences in the excitation intensity distribution along the depth direction on the surface of the sample. 
     The confocal microscope apparatus according to a third aspect of the present invention is characterized by comprising: a first optical system which illuminates a sample via an objective lens with incoherent light output from an incoherent light source, and detects fluorescence emitted from the sample via the objective lens; and a second optical scanning system which irradiates specific regions of a sample with a laser beam from a laser light source, thereby causing a particular phenomenon. The optical laser system and the microscope of Koehler illumination are combined, so that it becomes possible to change the width of measurement due to differences in the excitation intensity distribution along the depth direction on the surface of the sample. 
     Preferred embodiments of the confocal microscope described above are as described in the following. Each of the embodiments may be used alone or may used in combination. 
     (1) The second optical scanning system further comprises a beam diameter varying mechanism, which changes a beam diameter of the laser beam of the laser light source. 
     (2) An excitation light intensity distribution calculator which calculates and stores the excitation light intensity distribution along a depth direction on the sample surface from the beam diameter of the laser beam output from the beam diameter varying mechanism is further provided. 
     (3) The first laser light source is an IR pulsed laser, and the beam diameter varying mechanism is provided to the second scanning optical system. 
     (4) In (3), a depth direction intensity distribution calculator which calculates an intensity distribution along a depth direction of the laser light beam output from the beam diameter varying mechanism on the sample surface is further provided. 
     (5) The incoherent light source includes a lamp or an LED light source. 
     The observation method according to the fourth aspect of the invention is characterized by comprising: irradiating an excitation light to a sample to excite the sample; irradiating an light to cause the particular phenomenon to a desired position; and imaging by detecting a light from the excited sample, in which said irradiating the excitation light includes adjusting an intensity distribution of the excitation light along a depth direction on the surface. 
     The observation method according to the fifth aspect of the invention is characterized by comprising: irradiating an excitation light to a sample to excite the sample; irradiating an light to cause the particular phenomenon to a desired position; and imaging by detecting a light from the excited sample, in which said irradiating the sample includes adjusting an intensity distribution of the light to cause the particular phenomena along a depth direction on the surface. 
     The observation method according to the sixth aspect of the invention is characterized by comprising: irradiating an excitation light to a sample via a rotatable disk to acquire a fluorescent image of the sample by a disk scanning; and irradiating an light to cause the particular phenomenon to a desired position. With this configuration, it is preferable that the irradiating the light includes adjusting an intensity distribution of the excitation light along a depth direction on the surface. 
     According to the present invention, by independently changing the intensity distribution along the depth direction on the sample surface of the excitation light in the optical system for sample excitation and for obtaining images, it becomes possible to do dynamic analysis of different three-dimensional spaces. 
     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 inventive concept as defined by the appended claims and their equivalents.