Abstract:
A proposition is to accurately evaluate a phototoxic property (cell function) concerning a living cell. To this end, an evaluating method of cell function includes the operations of dyeing a specific site of the living cell with a fluorescent dye, irradiating the living cell with light to measure changes in brightness of resulting fluorescence generated at an adjacent site of the specific site, and evaluating the phototoxic property based on the brightness changes. In the event of functional depression in the specific site, the fluorescent dye cannot be retained therein and is extravasated into the adjacent site through the membrane of the specific site. The present embodiment can measure the extent of this extravasation, making it possible to accurately evaluate the phototoxic property.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application is a Continuation Application of International Application No. PCT/JP2007/000313, filed Mar. 28, 2007, designating the U.S., in which the International Application claims a priority date of Mar. 31, 2006, based on prior filed Japanese Patent Application No. 2006-098712, the entire contents of which are incorporated herein by reference. 
       BACKGROUND 
       [0002]    1. Field 
         [0003]    The present invention relates to an evaluating method of cell function, an evaluating system of cell function, a fluorescent microscope system, a photodynamic therapy method, and a photodynamic therapy system, applicable to photochemistry, photophysiology, photodynamic effects, and the like in life science. 2. Description of the Related Art 
         [0004]    Chromophore-assisted laser inactivation (CALI) and phot dynamic therapy (PDT) are among the techniques of photodynamic therapy. The former is the technique that suppresses activity of molecules, and the latter is the technique that induces damage to the cell membrane, organelles, and DNA to cause cell death. The letter technique, PDT, is effective in killing cancer cells. 
         [0005]    PDT works under the principle that a fluorescent dye in cells absorbs light and creates singlet oxygen and other chemical species, which are generated as by-products when the dye releases fluorescence. These chemical species would then damage part of cells to induce functional depression or cell death (see Patent Document 1 (Japanese Unexamined Patent Application Publication No. 2001-4542), Non-Patent Document 1 (Takato YOSHIDA, Eiji KAWANO, Takashi SAKURAI, kisojikken moderu kara rinshou deno PDT monitaringu no kanousei wo saguru, Nihon Laser Chiryou Gakkaishi, Vol. 2, No. 2, pp. 67-71, published on January, 2004), for example) 
         [0006]    In photodynamic therapy, in order to find therapeutic effects or side effects, it is important to accurately evaluate the damage (functional depression) incurred to the living cells by irradiation of light. In this specification, the term “phototoxic property” is used to describe the extent of functional depression incurred to the living cells by irradiation of light. 
         [0007]    Conventionally, phototoxic property has been evaluated only broadly based on whether cells have been killed by irradiation of light. In some techniques, the phototoxic property during light exposure is evaluated in real time by measuring the rate or extent of fluorescence bleaching. However, this method suffers from inaccuracy because the bleaching (decay of fluorescence brightness) is not well correlated with phototoxic property (functional depression of living cells). 
       SUMMARY 
       [0008]    Accordingly, it is a proposition of the present invention to provide an evaluating method of cell function, an evaluating system of cell function, and a fluorescent microscope system that are capable of accurately evaluating phototoxic property. Another proposition of the present invention is to provide a photodynamic therapy method capable of realizing an appropriate therapy, and a photodynamic therapy system suitable for such a photodynamic therapy method. 
         [0009]    An evaluating method of cell function of the present invention is a method of evaluating a cell function concerning a living cell, and the method includes dyeing operation of dyeing a specific site of the living cell with a fluorescent dye, measuring operation of measuring a brightness value of fluorescence generated at an adjacent site of the specific site as a result of irradiation of the stained living cell with light, and evaluating operation of evaluating the cell function based on changes in the measured brightness value. 
         [0010]    The fluorescent dye may be Rhodamin 123. 
         [0011]    Further, an evaluating method of cell function of the present invention may further include the operation of controlling the irradiation of light to the stained living cell at a predetermined timing according to the evaluation of cell function. 
         [0012]    Further, in the evaluating operation, the cell function may be evaluated based on a peak of a curve representing the brightness changes. 
         [0013]    Further, in the evaluating operation, the cell function may be evaluated based on an amount of light irradiation being spent until the brightness change curve reaches the peak. 
         [0014]    Further, in the evaluating operation, the cell function may be evaluated based on a brightness value at the peak of the brightness change curve. 
         [0015]    Further, the adjacent site of the specific site may be an area specified by a rectangular shaped frame, or an area specified by a closed and free curved frame or a closed and multiangular shaped frame in which the specific site is excluded from an area including the specific site and an area adjacent to the specific site. 
         [0016]    Further, the brightness value may be a maximum brightness value or a mean brightness value of a plurality of fluorescence brightness values measured in the adjacent site. 
         [0017]    Further, the specific site may be a mitochondrion, and the adjacent site may be a cellular cytoplasm. 
         [0018]    An evaluating system of cell function of the present invention is a system that evaluates a cell function concerning a living cell, the living cell including a specific site stained with a fluorescent dye in advance, and the evaluating system of cell function includes an irradiating unit that irradiates the stained living cell with light, a measuring unit that measures a brightness value of fluorescence generated at an adjacent site of the specific site as a result of the irradiation of light, and an evaluating unit that evaluates the cell function based on changes in the measured brightness value. 
         [0019]    The fluorescent dye may be Rhodamin 123. 
         [0020]    Further, an evaluating system of cell function of the present invention may further include a controlling unit that controls the irradiation of light to the stained living cell at a predetermined timing according to the evaluation of cell function. 
         [0021]    Further, the evaluating unit may evaluate the cell function based on a peak of a curve representing the brightness changes. 
         [0022]    Further, the evaluating unit may evaluate the cell function based on an amount of light irradiation being spent until the brightness change curve reaches the peak. 
         [0023]    Further the evaluating unit may evaluate the cell function based on a brightness value at the peak of the brightness change curve. 
         [0024]    Further, the adjacent site of the specific site may be an area specified by a rectangular shaped frame, or an area specified by a closed and free curved frame or a closed and multangular shaped frame in which the specific site is excluded from an area including the specific site and an area adjacent to the specific site. 
         [0025]    Further, the brightness value may be a maximum brightness value or a mean brightness value of a plurality of fluorescence brightness values measured in the adjacent site. 
         [0026]    A fluorescent microscope system of the present invention includes an excitation unit that irradiates a living cell with excitation light, an observing unit that acquires a fluorescence image of the living cell, and an evaluating system of cell function of the present invention, in which the excitation unit and the observing unit are also used as the irradiating unit and the measuring unit, respectively, of the evaluating system. 
         [0027]    Further, a photodynamic therapy method of the present invention is a method that irradiates a living cell with light, the method including dyeing operation of dyeing a specific site of the living cell with a fluorescent dye, therapeutic operation of irradiating the stained living cell with light, and evaluating operation of evaluating a cell function concerning the living cell by an evaluating method of cell function of the present invention. 
         [0028]    Further, a photodynamic therapy system of the present invention is a system that irradiates a living cell with light, the living cell including a specific site stained with a fluorescent dye in advance, the system including a therapeutic unit that irradiates the stained living cell with light, a measuring unit that measures a brightness value of fluorescence generated at an adjacent site of the specific site as a result of the irradiation of light, and a presenting unit that presents to an operator changes in the measured brightness value, in real time. 
         [0029]    The fluorescent dye may be Rhodamin 123. 
         [0030]    Further, the adjacent site of the specific site may be an area specified by a rectangular shaped frame, or an area specified by a closed and free curved frame or a closed and multiangular shaped frame in which the specific site is excluded from an area including the specific site and an area adjacent to the specific site. 
         [0031]    Further, the brightness value may be a maximum brightness value or a mean brightness value of a plurality of fluorescence brightness values measured in the adjacent site. 
         [0032]    The present invention realizes an evaluating method of cell function, an evaluating system of cell function, and a fluorescent microscope system that are capable of accurately evaluating phototoxic property. The invention also realizes a photodynamic therapy method capable of realizing an appropriate therapy, and a photodynamic therapy system suitable for such a photodynamic therapy method. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0033]      FIG. 1  is a diagram showing a configuration of a system of a First Embodiment. 
           [0034]      FIG. 2  is a diagram explaining fluorescence images I 1 , I 2 , . . . , I N . 
           [0035]      FIG. 3  is a flow chart representing an operation of a CPU  22  evaluating a phototoxic  10  property. 
           [0036]      FIG. 4  is a diagram explaining step S 1 . 
           [0037]      FIG. 5  is a diagram explaining steps S 2  and S 3 . 
           [0038]      FIG. 6  is a diagram showing changes in brightness of a stained site (mitochondria  41 ). 
           [0039]      FIG. 7  is a diagram showing a configuration of a system of a Second Embodiment. 
           [0040]      FIG. 8  is a fluorescence image obtained in an example. 
           [0041]      FIG. 9  is a graph of brightness values measured in the example. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS  
     First Embodiment 
       [0042]    The following will describe a First Embodiment of the present invention. The present embodiment embodies a confocal fluorescence microscope system with the function of evaluating a cell function. 
         [0043]    First, a configuration of the system is described. 
         [0044]      FIG. 1  is a diagram representing a configuration of the system. As shown in  FIG. 1 , the system includes a main body of microscope  10 , a computer  20 , and a monitor  30 , among other components. 
         [0045]    In the main body of microscope  10 , a specimen  17  is disposed that includes living cells. The specimen  17  has been supplemented with a fluorescent dye for mitochondria (for example, RH123: Rhodamin 123). The fluorescent dye stains only the mitochondria in the living cells, leaving the other organelles unstained. 
         [0046]    The main body of microscope  10  includes an excitation light source  11  that emits a laser beam. The laser beam includes at least a wavelength component that can serve as excitation light for the fluorescent dye (for example, a wavelength component of 507 nm). At least this wavelength component of the laser beam is reflected by a dichroic mirror  13  toward the specimen  17  through an optical scanner  15  and an objective lens  16 , and forms a single light spot on the specimen  17 . The fluorescent dye at this light spot generates fluorescence (529 nm), which is incident on the dichroic mirror  13  through the objective lens  16  and the optical scanner  15 . The fluorescence travels through the dichroic mirror  13  and falls on a pinhole mask  101  through an image-forming lens  19 . 
         [0047]    The pinhole mask  101  is conjugate to the specimen  17 , so that only the necessary light component of the fluorescence incident on the pinhole mask  101  passes through it. The fluorescence passing through the pinhole mask  101  enters a light sensor  102  where photoelectric conversion occurs. The fluorescence converted to an electrical signal in the light sensor  102  is then sent to the computer  20 . In the computer  20 , the electrical signal is converted to a digital signal and stored in a frame memory  21  of the computer  20 . 
         [0048]    In the main body of microscope  10 , the optical scanner  15  and the light sensor  12  are driven in synchronism to two-dimensionally scan the specimen  17  with a light spot, thereby repeatedly generating electrical signals. As a result, a fluorescence image of one frame is obtained from the specimen  17  (imaging of the specimen  17 ). The imaging magnifications of the objective lens  16  and the image-forming lens  19  are set to values suitable for the observation of microstructures (organelles) of the living cells. Accordingly, the fluorescence image contains one to several living cells. 
         [0049]    The main body of microscope  10  of the system repeats this imaging process N times, either continuously or intermittently, so that fluorescence images of N frames are obtained. For example, the imaging is repeated about 200 to 300 times (N≈200 to 300), with the scan rate of the light spot and the power of the excitation light source  11  maintained constant in each imaging. During a non-imaging period of each frame, no light is incident on the specimen  17 . Here, the number of imaging processes is proportional to the quantity of irradiated light on the specimen  17 . 
         [0050]    In each imaging, a CPU  22  in the computer  20  reads out digital signals accumulated in the frame memory  21  and creates a fluorescence image I of the specimen  17 . The fluorescence image I is stored in a hard disc drive  25 . After N times of imaging, fluorescence images I 1 , I 2 , . . . , I N  of N frames are accumulated in the hard disc drive  25 . As required, the fluorescence images I 1 , I 2 , . . . , I N  are output to the monitor  30  via an interface circuit  26 . 
         [0051]    The computer  20  also includes a ROM  23  and a RAM  24 , the former being a memory storing a basic operating program for the CPU  22 , and the latter a memory used in the operation of the computer  20  when needed. The hard disc drive  25  also stores a system operating program for the CPU  22 , which is read out at appropriate timings to cause the CPU  22  to perform various processes. In the system, the “evaluating processing of phototoxic property”, described later, is included in these processes. 
         [0052]      FIG. 2  shows an exemplary illustration of the fluorescence images I 1 , I 2 , . . . , I N  obtained in the system as above. In  FIG. 2 , the shades are lighter as the brightness diminishes. The subscript “i” appended to the fluorescence image I i  indicates a frame number, which is smaller for fluorescence images obtained earlier. 
         [0053]    As shown on the left in  FIG. 2 , in the first fluorescence image I 1 , mitochondria  41  at the stained site are the only bright component in a cell  40 , and no other components, including a cell nucleus  42  and other cell organelles (dotted portion) at the non-stained site appear in the image. A cellular cytoplasm  43  is completely dark. 
         [0054]    In the 50th fluorescence image I 50  shown in the middle in  FIG. 2 , the mitochondria  41  at the stained site appear darker as a result of bleaching, and the cellular cytoplasm  43  adjacent to the mitochondria  41  appears slightly brighter by fluorescing. 
         [0055]    This phenomenon is the indication of the functional depression of the mitochondria  41  incurred by the irradiation of light in the imaging and the resulting extravasation of the fluorescent dye into the cellular cytoplasm  43  from the inner side of the mitochondrial membrane. It should be noted here that not all fluorescent dyes in the mitochondria  41  extravasate to the cellular cytoplasm  43 . The extravasation stops at some time point. 
         [0056]    In the 200th fluorescence image I 200  shown on the right in  FIG. 2 , the cellular cytoplasm  43  appears dark by bleaching, and stably maintains a small brightness value as does the mitochondria  41 . 
         [0057]    As described, fluorescence occurs not only in the mitochondria  41  at the stained site but in the cellular cytoplasm  43  at the adjacent site in the cell  40 . This is related to the extravasation of the fluorescent dye from the mitochondria  41  to the cellular cytoplasm  43 , i.e., functional depression of the mitochondria  41 . 
         [0058]    By taking advantage of this, the system sets a reference point on the cellular cytoplasm  43 , and evaluates phototoxic property based on changes in brightness of the reference point. 
         [0059]    In the following, description is made as to the evaluating processing of phototoxic property performed by the CPU  22 . The process is performed after obtaining the fluorescence images I 1 , . . . , I N . 
         [0060]      FIG. 3  is a flow chart representing the operation of the CPU  22  evaluating a phototoxic property. As shown in  FIG. 3 , the CPU  22  performs a process for determining a reference point (step S 1 ), a process for referring to a brightness change of the reference point (step S 2 ), a process for calculating an evaluating value (step S 3 ), and a process for displaying the evaluating value (step S 4 ), in this order from the top. The following describes each step in order. 
       Step S 1  (Process for Determining Reference Point) 
       [0061]    In this step, as shown in  FIG. 4(A) , the CPU  22  refers to the first fluorescence image I 1  and compares the brightness value of each pixel of the fluorescence image I 1  with a threshold value to find pixels whose brightness values exceed the threshold value. The area where such pixels reside is regarded as a stained area  44 A. Since the only fluorescing component in the first fluorescence image I 1  is the mitochondria  41 , the area containing the mitochondria  41  is regarded as the stained area  44 A. 
         [0062]    The CPU  23  then sets a reference point  40 P at coordinates separated from the stained area  44 A by a small distance represented by predetermined coordinates. The predetermined coordinates have been set to appropriate values to locate the reference point  40 P on the cellular cytoplasm  43 . 
       Step S 2  (Process for Referring to Brightness Change of the Reference Point) 
       [0063]    In this step, the CPU  22  extracts brightness values P 1 , . . . , P N  of the reference point  40 P from the fluorescence images I 1 , . . . , I N . The subscript “i” appended to the brightness value P indicates the frame number. These brightness values P 1 , . . . , P N  may come from a single pixel at the reference point  40 P, or preferably from a plurality of pixels (pixels in an arbitrarily-shaped area) at the reference point  40 P, in which case a mean brightness value or maximum brightness value of the pixels is used as the brightness value P 1 , . . . , P N .  FIG. 4(B)  will be described later. 
         [0064]      FIG. 5(A)  shows a graph plotting the brightness values P 1 , . . . , P N  of the reference point  40 P, in which the horizontal axis represents the frame number, and the vertical axis represents the brightness value. From these data, the CPU  22  finds changes in brightness of the reference point  40 P. 
         [0065]    As shown in  FIG. 5(A) , the brightness value of the reference point  40 P increases as the frame number increases (increase in an amount of light irradiation), and reaches a peak in a certain frame (the 50th frame in the figure). The brightness value then decreases until it stabilizes at low brightness values in certain frames (around the 100th frame in the figure). The rate of increase of the brightness value indicates the chromogenic rate of the cellular cytoplasm  43 , i.e., the functional depression rate of the mitochondria  41 . 
         [0066]    Here, when the frame number at which the brightness value has the peak is f, the frame number f becomes smaller as the functional depression rate of the mitochondria  41  becomes faster, and larger as the functional depression rate of the mitochondria  41  becomes slower. To test this, the functional depression rate was slowed by reducing the power of the excitation light source  11 , with the other conditions held constant. As expected, the frame number f increased, as shown in  FIG. 5(B) . 
         [0067]    Step S 3  (Process for Calculating Evaluating Value) 
         [0068]    In this step, the CPU  22  calculates a frame number f ( 50  in  FIG. 5(A) ) at which the brightness value of the reference point  40 P has the peak, as shown by the arrow in  FIG. 5(A) . Based on this frame number f, the CPU  22  calculates an evaluating value E of phototoxic property. The evaluating value E is given by, for example, E=1/f, E=α/f, E=f A −f, or E=f A −αf (where α and f A  are constants), so that larger evaluating values E are obtained as the functional depression rate of the mitochondria  41  becomes faster. 
         [0069]    Step S 4  (Process for Displaying Evaluating Value) 
         [0070]    In this step, the CPU  22  displays the calculated evaluating value E on the monitor  30 . Here, it is preferable that the CPU  22  display the current fluorescence image IN along with the evaluating value E, and, as a marker for an operator, a mark such as a crosshair cursor or a rectangular shaped frame superimposed on the reference point  40 P. 
         [0071]    As described, the system evaluates phototoxic property through repeated imaging of the specimen  17  performed by the main body of microscope  10 . The evaluation is performed based on brightness changes ( FIG. 5 ) at the adjacent site (here, the cellular cytoplasm  43 ) of the stained site (here, the mitochondria  41 ), which is referred to instead of the brightness changes at the stained site. This evaluation yields proper results because the brightness changes at the adjacent site (here, the cellular cytoplasm  43 ) are well correlated with the functional depression at the stained site (here, the mitochondria  41 ), as described above. 
         [0072]    Further, in the evaluation performed by the system, because the frame number f at which the brightness value at the adjacent site (here, the cellular cytoplasm  43 ) has the peak is reflected in the evaluating value E, the evaluating value E accurately reflects the chromogenic rate of the adjacent site (here, the cellular cytoplasm  43 ) or the functional depression rate of the stained site (here, the mitochondria  41 ). That is, the evaluating value E is an accurate indication of phototoxic property. 
         [0073]    For comparison,  FIG. 6(A)  and  FIG. 6(B)  show brightness changes at the stained site (here, the mitochondria  41 ).  FIG. 6(A)  represents a graph obtained with the excitation light source  11  held at high power, and  FIG. 6(B)  represents a graph obtained with the excitation light source  11  held at low power. As shown in  FIG. 6(A)  and  FIG. 6(B) , the brightness change curves obtained from the stained site (here, the mitochondria  41 ) at different power levels of the excitation light source  11  have the peaks at the same point (frame number  1 ). It is therefore difficult to calculate an evaluating value of phototoxic property from these brightness change curves. 
         [0074]    Further, as described, while the brightness change curve from the stained site (here, the mitochondria  41 ) shows there is bleaching at the stained site (here, the mitochondria  41 ), it does not necessarily mean there is functional depression. As such, the evaluating value of phototoxic property calculated from this brightness change curve would not be as accurate as the evaluating value E obtained in this embodiment. 
       Others 
       [0075]    In this system, it is preferable that the mathematical formula deriving the evaluating value E from the frame number f be appropriately formulated such that the actual phototoxic property and the evaluating value E are linearly related to each other. This is possible by experiments or simulations using systems including the living cell in which the phototoxic property is known. 
         [0076]    Further, in this system, the evaluating value E of phototoxic property is defined by the frame number f at the peak brightness value. However, the evaluating value E may be defined by the brightness value (peak brightness value) when the brightness has the peak. Further, the evaluating value E may also be defined by both the frame number f and the peak brightness value. 
         [0077]    The foregoing description of the present embodiment was given through the case where fluorescence images of about 200 frames were obtained, in order to illustrate the evaluation of phototoxic property in the stained area  44 A using changes in brightness value of the reference point  40 P. However, from the standpoint of preventing unnecessary damage to the cells, it is preferable that the irradiation of a laser beam from the excitation light source  11  be stopped or the intensity of the laser beam be reduced immediately after the brightness value of the reference point  40 P has reached the peak and starts to decline, or after a predetermined period of time (several seconds) has elapsed from such an event. 
         [0078]    Because the system uses a confocal microscope as the main body of microscope  10 , a plurality of fluorescence images with different sectionings can be obtained. Such multiple fluorescence images can be used to improve the accuracy of evaluating value E. 
         [0079]    The present embodiment has been described through the case where the stained site is mitochondria  41 ; however, other organelles or the area outside of the cell membrane (culture fluid) may be used as the stained site. When the stained site is the cell nucleus, the reference point may be set on the cellular cytoplasm  43 . When the stained site is the cellular cytoplasm  43 , the reference point may be set on the culture fluid. Further, when the stained site is the culture fluid, the reference point may be set on the cellular cytoplasm  43 . In any case, the reference point is set at a site adjacent to the stained site with a membrane in between. 
         [0080]    In the foregoing description, the computer  20  performs each process of the system. However, the operation of the computer  20  may be executed either partially or entirely by a device (control device, image processing device) designated to the main body of microscope  10 , or by an operator. 
         [0081]    For example, the reference point  40 P, which was described as being automatically decided by the computer  20  in the system, may be entered by an operator through an input device (keyboard, mouse, or the like; not shown). When an operator is allowed to enter the reference point  40 P, a marker may be superimposed on the fluorescence image  11  displayed on the monitor  30  as shown in  FIG. 4(A)  and  FIG. 4(B) . The marker may be a crosshair cursor  ( FIG. 4(A) ) or a rectangular shaped frame. Alternatively, the marker may be an arbitrarily-shaped frame, such as a closed and free curved frame or a closed and multiangular shaped frame, that is displayed to exclude the stained area  44 A (mitochondria  41 ) from the area including the stained area  44 A and the cellular cytoplasm  43  adjacent thereto ( FIG. 4(B) ). In the example shown in  FIG. 4(B) , the area defined by the annular frame is the reference point  40 P. The arbitrarily-shaped frame defining the reference point  40 P may be variable in size. 
         [0082]    The main body of microscope  10  of the system, which has been described as a microscope that obtains fluorescence images, may be modified to obtain both fluorescence images and differential interference images. In this case, the differential interference image may be superimposed on the fluorescence image displayed on the monitor  30 . The superimposed differential interference image allows for observation of non-fluorescing organelles (transparent organelles). Further, the differential interference image can be used to set the reference point. In this case, failure to set the reference point becomes less likely. 
         [0083]    Further, in this system, the operating program for the CPU  22  has been described as being pre-stored in the hard disc drive  25 . However, the program may be installed either partially or entirely in the computer  20  via, for example, the Internet or CD-ROM (not shown). 
         [0084]    Further, the main body of microscope  10  of the system, described as a confocal microscope that detects a confocal point of the light from the specimen  17 , may omit this function. In this case, the pinhole mask  101  will not be required. Further, the main body of microscope  10  may be modified to a multiphoton microscope that attains the confocal effect by methods other than using the pinhole mask. 
         [0085]    Further, the main body of microscope  10  of the system, which is a scanning microscope for scanning the specimen  17  with light, may be a non-scanning microscope when it omits the confocal point detecting function. In this case, the optical scanner  15  will not be required, and an imaging sensor is provided instead of the light sensor  102 . 
         [0086]    Further, the system is applicable to evaluation of cell function using a drug, by supplying a drug to the mitochondria  41  with the fluorescent dye. Further, the system is applicable to evaluation of cell function by heat or radiation, by applying heat or radiation with light. 
       Second Embodiment 
       [0087]    The following will describe a Second Embodiment of the present invention. The present embodiment embodies a photodynamic therapy system, and a photodynamic therapy method using it. 
         [0088]      FIG. 7  is a diagram showing a configuration of the present system. As shown in  FIG. 7 , the system is primarily made up of four components, including a therapeutic objective ST 1 , a therapeutic system ST 2 , an observing system ST 3 , and an excitation system ST 4 . 
         [0089]    The therapeutic objective ST 1  is, for example, an affected area including cancer cells, supplemented beforehand with a fluorescent dye for mitochondria (for example, RH123). The fluorescent dye is used for the evaluation of phototoxic property (evaluation of therapeutic effect). 
         [0090]    The therapeutic system ST 2  irradiates the therapeutic objective ST 1  with radiation rays (such as gamma rays) or laser light for therapy (ultraviolet range, visible range, infrared range), so as to induce cell injury or cell death in cancer cells. The gamma rays have the effect of solely inducing cell injury, while the laser light for therapy induces cell injury (or cell death) by reacting with the fluorescent dye applied to the therapeutic objective ST 1 . The following describes the case using the latter (PDT). 
         [0091]    The laser light for therapy is generated in a radiation device  51  provided in the therapeutic system ST 2 , and is emitted as pulsed oscillations toward the therapeutic objective ST 1 , from a tube tip (head)  52 , measuring several millimeters to several centimeters in diameter, provided at the tip of the therapeutic system ST 2 . The head  52  is provided to improve the efficiency of concentrating the energy of the laser light for therapy onto the therapeutic objective ST 1 . 
         [0092]    The excitation system ST 4  includes an excitation light source  11  and a dichroic mirror  13 . Through an objective lens  16  of the observing system ST 3 , the excitation system ST 4  emits excitation light (for example, a wavelength of 507 nm) as pulsed oscillations toward the therapeutic objective ST 1 . The excitation light is emitted alternately with the laser light for therapy. 
         [0093]    The observing system ST 3  includes the objective lens  16 , an image-forming lens  19 , an imaging sensor  102 ′, a circuit part  20 ′, and a monitor  30 , among others. The fluorescence generated in the therapeutic objective ST 1  during the irradiation of the excitation light is captured by the objective lens  16  and the image-forming lens  19  of the observing system ST 3 , and a fluorescence image of the therapeutic objective ST 1  is formed on the imaging sensor  102 ′. The imaging sensor  102 ′ continuously captures the fluorescence images, which are then output to the monitor  30  one after another via the circuit part  20 ′. 
         [0094]    The imaging magnifications of the objective lens  16  and the image-forming lens  19  of the observing system ST 3  are set to values suitable for the observation of microstructures (organelles) of the cells. Accordingly, cells  40  of the therapeutic objective ST 1  are displayed in real time on the monitor  30 . 
         [0095]    It should be noted here that the head of the observing system ST 3  and the excitation system ST 4 , and the head  52  of the therapeutic system ST 2  are facing substantially the same point on the therapeutic objective ST 1 , so that the imaging point of the fluorescence image substantially coincides with the irradiation point of the laser light for therapy. To suppress any misregistration between the two, these heads may be fixed or the same head may be used. 
         [0096]    During a course of therapy, an operator observes the stained site (here, the mitochondria  41 ) and the adjacent site (here, the cellular cytoplasm  43 ) on the monitor  30  while the therapeutic objective ST 1  is being irradiated with the laser light for therapy. Here, the operator looks at the brightness of the adjacent site (here, the cellular cytoplasm  43 ) and evaluates the phototoxic property (therapeutic effect) of the therapeutic system ST 2  according to the timing at which the brightness reaches the peak, or the extent of brightness when it has the peak. According to the result of evaluation, the operator suspends the irradiation of the laser light for therapy at an appropriate timing, or adjusts the power of the therapeutic system ST 2  at an appropriate level. 
         [0097]    In this manner, the system allows the operator to perform photodynamic therapy while evaluating the therapeutic effect in real time, making it possible to perform an appropriate therapy without failing to remove cancer tissues by underexposure of the laser light for therapy, or without causing any side effect by overexposure of the laser light for therapy. 
         [0098]    While the system was described in which the operator visually checks the brightness of the adjacent site (here, the cellular cytoplasm  43 ), the brightness may be checked by automation. In this case, the circuit part  20 ′ of the observing system ST 3  extracts a brightness signal of the adjacent site (here, the cellular cytoplasm  43 ) from the output of the imaging sensor  102 ′, and notifies the operator of the level of the brightness signal in real time. The notification may be given on the monitor  30 , or by playing sounds from a separately provided sound output device (speaker). 
         [0099]    Further, while the excitation light and the light for therapy are separately provided in the system described above, the light for therapy may be used to also provide the excitation light, when it contains a wavelength component for the excitation light. 
         [0100]    The therapeutic apparatus described in this Second Embodiment can be made into a diagnostic apparatus simply by replacing the therapeutic system ST 2  with a diagnostic system. The diagnostic system includes a diagnostic wave (sound wave, electromagnetic wave) generator, an illuminating optical system for illuminating the affected area, an imaging sensor, and an imaging optical system for condensing the reflected light from the illuminated affected area onto the imaging sensor. 
       EXAMPLE 
       [0101]    The following describes an example of the evaluation of cell function according to the present invention, performed with the confocal fluorescence microscope system of the First Embodiment. 
         [0102]    RH123, used as a fluorescent dye, was applied to the mitochondria in living cells to prepare a specimen. The specimen was two-dimensionally scanned by irradiating an argon laser (488 nm) emitted in a predetermined intensity from the excitation light source  11 , so as to obtain a fluorescence image of one frame. 
         [0103]      FIG. 8(A)  is the actual first fluorescence image obtained. Predetermined positions of the mitochondria (stained area) and the cellular cytoplasm (reference point) were specified by rectangular shaped frames, and a mean value of brightness values of the pixels in each of these specific areas was determined. These mean values were used as the brightness value of the mitochondria, and the brightness value of the cellular cytoplasm, respectively. 
         [0104]    This procedure of obtaining the fluorescence image was repeated under the same conditions, and the brightness values of the mitochondria and the cellular cytoplasm were measured.  FIG. 8(B)  is the actual 50th fluorescence image, and  FIG. 8(C)  is the actual 150th fluorescence image obtained. It can be seen from  FIG. 8  that the fluorescence image indeed undergoes changes as described in the First Embodiment with reference to  FIG. 2 . 
         [0105]      FIG. 9(A)  is the actual graph plotting the brightness values of the mitochondria and the cellular cytoplasm measured by the irradiation of an argon laser of a predetermined intensity. In  FIG. 9(A) , the decrease in the brightness value of the mitochondria is due to bleaching and a release of RH123 into the cellular cytoplasm. The increase of the brightness value of the cellular cytoplasm is due to the RH123 released by the mitochondria, and the peak indicates the end of the RH123 release into the cellular cytoplasm. That is, the graph tells that the functional depression of the mitochondria is complete when the image with the frame number  50  is obtained, in which the brightness value of the cellular cytoplasm has the peak. 
         [0106]    The brightness values of the mitochondria and the cellular cytoplasm were also measured under the same conditions except for reducing the intensity of the laser beam emitted by the excitation light source  11 .  FIG. 9(B)  is the actual graph plotting the brightness values of the mitochondria and the cellular cytoplasm measured by the irradiation of a laser beam having a weaker intensity than the laser used in  FIG. 9(A) . As can be seen in  FIG. 9(B) , the brightness value of the cellular cytoplasm has the peak in frame number  70 , showing that the functional depression of the mitochondria proceeds at a slower rate when the intensity of the excitation laser beam is weaker. 
         [0107]    The many features and advantages of the embodiments are apparent from the detailed specification and, thus, it is intended by the appended claims to cover all such features and advantages of the embodiments that fall within the true spirit and scope thereof. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the inventive embodiments to the exact construction and operation illustrated and described, and accordingly all suitable modifications and equivalents may be resorted to, falling within the scope thereof.