Abstract:
A scanning fluorescent microscope includes a light source which irradiates a sample with an exciting light pulse, and a splitter which splits fluorescent photons emitted from the sample excited by the exciting light pulse, in at least first and second groups. The scanning fluorescent microscope also includes first and second detectors which detect the fluorescent photons of the first and second group, respectively, first and second counters which count numbers of fluorescent photons detected by the detectors, respectively, first and second correcting units which perform a predetermined correction to the numbers of fluorescent photons, an adder adding the numbers of fluorescent photons corrected, and an operating unit which calculates a florescent lifetime based on resulting numbers from the adder.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
   This application is based upon and claims the benefit of priority from Japanese Patent Application No. 2004-115681, filed Apr. 9, 2004, the entire contents of which are incorporated herein by reference. 
   This application is related to the following commonly assigned applications: U.S. Ser. No. 10/769,135 filed Jan. 30, 2004; U.S. Ser. No. 10/741,522 filed Dec. 18, 2003, all of which are incorporated herein by reference. 
   BACKGROUND OF THE INVENTION 
   1. Field of the Invention 
   The present invention relates to a scanning fluorescent microscope that irradiates a sample with exciting light pulses, counts the number of fluorescent photons emitted from the excited sample, and measures at least a fluorescent lifetime based on the counted number of the fluorescent photons. 
   2. Description of the Related Art 
   To examine the excited state of a sample, a conventionally known technique includes irradiating the sample with exciting light to turn the sample into an excited state, measuring the fluorescence emitted from the sample during the transition from the excited state to the ground state, and calculating the fluorescent lifetime. One known method of calculating a fluorescent lifetime is a Time-Gate Technique, according to which a sample is irradiated with exciting light pulses, the number of fluorescent photons emitted from the sample is measured at plural periods of time, and the fluorescent lifetime is calculated based on the measured numbers of fluorescent photons (see C. J. DE GRAUW and H. C. GERRITSEN, “Multiple Time-Gate Module for Fluorescence Lifetime Imaging”, APPLIED SPECTROSCOPY, Volume 55, Number 6, 2001). 
   In order to minimize the error in fluorescent lifetime calculation, measurement should be conducted on a large number of fluorescent photons. However, an increase in the emission rate of fluorescent photons with an increase in the intensity of the exciting light results in an increase in the measurement error of the lifetime. This is because that the fluorescent photons successively entering a measurement device cannot be captured within the measurement resolution thereof. Hence, for the minimization of the error in fluorescent lifetime calculation, the number of emitted fluorescent photons per one irradiation of exciting light (emission rate) needs to be equal to or less than 0.01. In other words, calculation of fluorescent lifetime is extremely time-consuming when error minimization is required. In addition, in calculation of the fluorescent lifetime of a living body, for example, in which the fluorescent lifetime changes over time, the speed of calculation cannot outpace the speed of changes over time, whereby the observation of fluorescent lifetime distribution in a living body or the like is virtually impossible. 
   SUMMARY OF THE INVENTION 
   A scanning fluorescent microscope according to one aspect of the present invention includes a light source irradiating a sample with an exciting light pulse, and a splitter splitting fluorescent photons emitted from the sample excited by the exciting light pulse, in at least a first group and a second group. The scanning fluorescent microscope also includes a first detector detecting the fluorescent photons of the first group, a second detector detecting the fluorescent photons of the second group, a first counter counting a number of fluorescent photons detected by the first detector, a second counter counting a number of fluorescent photons detected by the second detector, a first correcting unit performing a predetermined correction to the number of fluorescent photons counted by the first counter, a second correcting unit performing a predetermined correction to the number of fluorescent photons counted by the second counter, an adder adding the numbers of fluorescent photons corrected by the first and second correcting units, and an operating unit calculating a florescent lifetime based on resulting numbers from the adder. 
   A scanning fluorescent microscope according to another aspect of the present invention includes a light source irradiating a sample with an exciting light pulse, a polarization splitter splitting fluorescent photons emitted from the sample excited by the exciting light pulse, in at least a first polarization component and a second polarization component. The scanning fluorescent microscope also includes a first detector detecting the fluorescent photons of the first polarization component, a second detector detecting the fluorescent photons of the second polarization component, a first counter counting a number of fluorescent photons detected by the first detector, a second counter counting a number of fluorescent photons detected by the second detector, a first correcting unit performing a predetermined correction to the number of fluorescent photons counted by the first counter, a second correcting unit performing a predetermined correction to the number of fluorescent photons counted by the second counter, and a polarization operating unit obtaining an orientation of the sample based on the numbers of fluorescent photons corrected by the first and second correcting units. 
   A scanning fluorescent microscope according to still another aspect of the present invention includes a light source irradiating a sample with an exciting light pulse, a splitter splitting fluorescent photons emitted from the sample excited by the exciting light pulse, in at least a first group and a second group, and a polarization splitter splitting the fluorescent photons of the first group in at least a first polarization component and a second polarization component. The scanning fluorescent microscope includes a first detector detecting the fluorescent photons of the first polarization component, a second detector detecting the fluorescent photons of the second polarization component, a first counter counting a number of fluorescent photons detected by the first detector, a second counter counting a number of fluorescent photons detected by the second detector, a first correcting unit performing a predetermined correction to the number of fluorescent photons counted by the first counter, a second correcting unit performing a predetermined correction to the number of fluorescent photons counted by the second counter, and a polarization operating unit obtaining an orientation of the sample based on the numbers of fluorescent photons corrected by the first and second correcting units. 
   The above and other objects, features, advantages and technical and industrial significance of this invention will be better understood by reading the following detailed description of presently preferred embodiments of the invention, when considered in connection with the accompanying drawings. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a schematic block diagram of a scanning fluorescent microscope according to a first embodiment of the present invention; 
       FIG. 2  is a detailed block diagram of a controller according to the first embodiment of the present invention; 
       FIG. 3  is a timing chart of transition of a laser source and a switch between an “ON” state and an “OFF” state according to the first embodiment of the present invention; 
       FIG. 4  is a schematic block diagram of a scanning fluorescent microscope according to a modification of the first embodiment of the present invention; 
       FIG. 5  is a schematic block diagram of a scanning fluorescent microscope according to a second embodiment of the present invention; 
       FIG. 6  is a schematic block diagram of a scanning fluorescent microscope according to a modification of the second embodiment of the present invention; and 
       FIG. 7  is a schematic block diagram of a scanning fluorescent microscope according to a third embodiment of the present invention. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
   Exemplary embodiments of a scanning fluorescent microscope according to the present invention will be explained in detail below with reference to the accompanying drawings. 
     FIG. 1  is a block diagram of a schematic structure of a scanning fluorescent microscope  100  according to a first embodiment of the present invention. In  FIG. 1 , the scanning fluorescent microscope  100  includes a laser source  1 , a collimating lens  2 , a dichroic mirror  3 , a galvanometer mirror  4 , a pupil projection lens  5 , a mirror  6 , an observation lens barrel  7 , an imaging lens  8 , an observation lighting unit  9 , a half mirror  10 , an objective lens  11 , a sample  12 , a measuring unit  13 , a controller  20 , and a display  25 . 
   The measuring unit  13  includes a condenser lens  14 , a pinhole  15 , a half mirror  16 , absorption filters  17   a  and  17   b , and photodetectors  19   a  and  19   b . The controller  20  includes signal processing units  21   a  and  21   b , correcting units  22   a  and  22   b , an adder  23 , and an operating unit  24 . 
   Under the control by the controller  20 , exciting light pulses are emitted from the laser source  1 . The exciting light pulse is reflected by the dichroic mirror  3  via the collimating lens  2  to sequentially pass through the galvanometer mirror  4 , the pupil projection lens  5 , the mirror  6 , the observation lens barrel  7 , the imaging lens  8 , the half mirror  10 , and the objective lens  11 , to be collected on the sample  12 . The exciting light pulse thus focused on the sample excites the sample  12  to cause emission of fluorescent photons from the sample  12 . The fluorescent photons emitted from the sample  12  move the optical path of the exciting light pulse backward from the objective lens  11  up to the dichroic mirror  3  and then pass through the dichroic mirror  3  to enter the measuring unit  13 . After entering the measuring unit  13 , the fluorescent photons sequentially pass through the condenser lens  14  and the pinhole  15  to enter the half mirror  16 . The half mirror  16  reflects incoming fluorescent photons with the probability of 50% and transmits incoming fluorescent photons with the probability of 50%. For example, when one thousand fluorescent photons enter the half mirror  16 , ideally five hundred fluorescent photons are reflected by the half mirror  16  to enter the photodetector  19   a  via the absorption filter  17   a . The photodetector  19   a  outputs an electric signal Sa to the controller  20  for every incoming fluorescent photon. On the other hand, the remaining five hundred fluorescent photons not reflected by the half mirror  16  pass through the half mirror  16  to enter the photodetector  19   b  via the absorption filter  17   b . The photodetector  19   b  outputs an electric signal Sb to the controller  20  for every incoming fluorescent photon. 
   The capacity of each of photodetectors  19   a  and  19   b  is restricted so that the emission rate of the fluorescent photons per irradiation would be equal to or less than 0.01 as described above, when a conventional Time-Correlated Single Photon Counting (TCSPC) is employed for the calculation of fluorescent lifetime without correction of the counted number of fluorescent photons. However, the detection of the fluorescent photons with the half mirror  16  and two photodetectors  19   a  and  19   b  allows the emission rate up to 0.02. 
   The signal processing units  21   a  and  21   b  in the controller  20  count the number of incoming electric signals Sa and Sb, respectively, at plural time gates which are set at various time period, to supply the counted numbers of fluorescent photons to the correcting units  22   a  and  22   b , respectively. The correcting units  22   a  and  22   b  correct the received numbers of fluorescent photons in a predetermined manner to supply the resulting numbers to the adder  23 . The adder  23  adds the corrected numbers of fluorescent photons and provides the resulting number of fluorescent photons to the operating unit  24 . The operating unit  24  calculates the fluorescent lifetime based on the received sum of the numbers of fluorescent photons to supply the fluorescent lifetime to the display  25 . The display  25  converts the received fluorescent lifetime into a display signal to output for display. 
   Next, the correcting operation by the correcting units  22   a  and  22   b  will be described with reference to  FIG. 2 .  FIG. 2  is a block diagram of a schematic structure of the controller  20 . The controller  20  includes the signal processing units  21   a  and  21   b , the correcting units  22   a  and  22   b , the adder  23 , and the operating unit  24 . Further, the signal processing unit  21   a  includes switches SW 1   a  and SW 2   a , waveform discriminators  211   a  and  212   a , and counters  213   a  and  214   a , whereas the signal processing unit  21   b  includes switches SW 1   b  and SW 2   b , waveform discriminators  211   b  and  212   b , and counters  213   b  and  214   b.    
   While the photodetector  19   a  supplies the electric signal Sa to the signal processing unit  21   a , the photodetector  19   b  supplies the electric signal Sb to the signal processing unit  21   b . The timing of switching over between the “ON” state and the “OFF” state by the switches SW 1   a  and SW 1   b  is controlled in accordance with gate control signals from the controller  20 . Only in the “ON” operation, the electric signals Sa and Sb are supplied to the counters  213   a  and  213   b  via the waveform discriminators  211   a  and  211   b . The counters  213   a  and  213   b  count and output the numbers of fluorescent photons k 1   a  and k 1   b  to the correcting units  22   a  and  22   b , respectively. The timing of switching over between the “ON” state and the “OFF” state by the switches SW 2   a  and SW 2   b  is controlled in accordance with gate control signals from the controller  20 . Only in the “ON” operation, the electric signals Sa and Sb are supplied to the counters  214   a  and  214   b  via the waveform discriminators  212   a  and  212   b , respectively. The counters  214   a  and  214   b  count and output the numbers of fluorescent photons k 2   a  and k 2   b , to the correcting units  22   a  and  22   b , respectively. 
   The correcting unit  22   a  corrects the received numbers of fluorescent photons k 1   a  and k 2   a  to numbers of fluorescent photons m 1   a  and m 2   a  to be supplied to the adder  23 , whereas the correcting unit  22   b  corrects the received numbers of fluorescent photons k 1   b  and k 2   b  to numbers of fluorescent photons m 1   b  and m 2   b  to be supplied to the adder  23 . The adder  23  includes adders  23   a  and  23   b . The adder  23   a  adds the numbers of fluorescent photons m 1   a  and m 1   b  to output the resulting number of fluorescent photons m 1  (=m 1   a +m 1   b ) to the operating unit  24 , whereas the adder  23   b  adds the numbers of fluorescent photons m 2   a  and m 2   b  to output the resulting number of fluorescent photons m 2  (=m 2   a +m 2   b ) to the operating unit  24 . The operating unit  24  calculates the fluorescent lifetime based on the received numbers of fluorescent photons m 1  and m 2 . 
   The correcting operation by the correcting units  22   a  and  22   b  will be described. In general, the emission rate of the fluorescent photons in the transition process of the sample  12  from the excited state to the ground state corresponds with the Poisson distribution. With an increase in the energy of exciting light, the emission rate of the fluorescent photons increases, and successive emission of fluorescent photons occurs, which tends to result in an increase in the measurement error of the number of fluorescent photons. However, if the measured number of fluorescent photons obtained in a state of high emission rate is corrected according to the Poisson distribution, the fluorescent lifetime can be calculated with little error. The correcting units  22   a  and  22   b  correct the measured numbers of fluorescent photons according to the Poisson distribution. 
   Assume that “μ” represents the average number of incoming fluorescent photons among the fluorescent photons emitted from the sample  12  during a predetermined time gate ΔT per one irradiation of exciting light, and that “p (r, μ)” represents the probability of the number of incoming fluorescent photons during the time gate ΔT being r. Then, the probability p(r, μ) can be expressed by Equation (1): 
                   p   ⁡     (     r   ,   μ     )       =         ⅇ     -   μ       ·     μ   r         r   !               (   1   )               
Provided that r is equal to or larger than 1, Equation (1) leads to Equation (2).
   p ( r≧ 1,μ)=1− p (0,μ)=1− e   −μ   (2) 
Provided that “N” represents the number of irradiations of exciting light, and “k” represents the number of fluorescent photons measured at the time gate ΔT, the average number “x” of fluorescent photons measured at the time gate ΔT (the number also referred to as ‘count rate’) can be expressed by “x=k/N”. On the other hand, the probability p(0, μ) that the fluorescent photons are not incident at the time gate ΔT can be expressed by Equation (3) where “x” represents the count rate:
   p (0,μ)=( N−k )/ N= 1− x   (3) 
Similarly, the probability p(0, μ) that the fluorescent photons are not incident at time gate ΔT can be expressed by Equation (4) which is derived from Equation (1):
   p (0,μ)= e   −μ   (4) 
Hence, based on Equations (3) and (4), the average number μ of incoming fluorescent photons at the time gate ΔT can be expressed by Equation (5):
 μ=− In (1− x )  (5) 
Here, provided that “m” represents the number of incoming fluorescent photons at the time gate ΔT, “m” can be expressed as the multiplication of “N” and “μ”, where “N” is the number of irradiations of the exciting light and “μ” is the average number of incoming fluorescent photons at the time gate ΔT.
 
   In other words, the correcting units  22   a  and  22   b  solve Equation (6) for “N” which is the number of irradiations by the laser source  1  and “k” which is the measured number of fluorescent photons input from the signal processing units  21   a  and  21   b , and correct the measured number of fluorescent photons k to the number of actually incoming fluorescent photons m. 
   
     
       
         
           
             
               
                 m 
                 = 
                 
                   
                     
                       ∑ 
                       
                         r 
                         = 
                         1 
                       
                       ∞ 
                     
                     ⁢ 
                     
                       r 
                       · 
                       
                         p 
                         ⁡ 
                         
                           ( 
                           
                             r 
                             , 
                             μ 
                           
                           ) 
                         
                       
                       · 
                       N 
                     
                   
                   = 
                   
                     μ 
                     · 
                     N 
                   
                 
               
             
             
               
                 ( 
                 6 
                 ) 
               
             
           
         
       
     
   
   The calculating operation by the operating unit  24  will be described. Δ first time gate ΔT 1  and a second time gate ΔT 2  are set as plural time gates. Provided that “k 1 ” represents the measured number of fluorescent photons at the first time gate ΔT 1 , “k 2 ” represents the measured number of fluorescent photons at the second time gate ΔT 2 , “t” represents the time difference between the start of the measurements for the first time gate ΔT 1  and the second time gate ΔT 2 , and that the length of the first time gate ΔT 1  is equal to the length of the second time gate ΔT 2 , the fluorescent lifetime τ can be expressed by Equation (7): 
   
     
       
         
           
             
               
                 τ 
                 = 
                 
                   t 
                   
                     In 
                     ⁡ 
                     
                       ( 
                       
                         k 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         
                           1 
                           / 
                           k 
                         
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         2 
                       
                       ) 
                     
                   
                 
               
             
             
               
                 ( 
                 7 
                 ) 
               
             
           
         
       
     
   
     FIG. 3  is a timing chart of the timing of transition between the “ON” operation and the “OFF” operation of the laser source  1  and of the switches SW 1   a , SW 1   b , SW 2   a , and SW 2   b . The controller  20  sets the time difference t of the first time gate ΔT 1  and the second time gate ΔT 2  through the control of the timing of incidence of the laser beam from the laser source  1  and the switches SW 1   a , SW 1   b , SW 2   a , and SW 2   b.    
   The correcting unit  22   a  corrects the measured numbers of fluorescent photons k 1   a  and k 2   a  to the numbers m 1   a  and m 2   a  through the calculation of Equation (6) to output to the adder  23 , whereas the correcting unit  22   b  corrects the measured numbers of fluorescent photons k 1   b  and k 2   b  to the numbers m 1   b  and m 2   b  through the calculation of Equation (6) to output to the adder  23 . The adder  23   a  in the adder  23  adds the numbers of fluorescent photons m 1   a  and m 1   b  to output the number of incoming fluorescent photons m 1  (=m 1   a +m 1   b ) at the first time gate ΔT 1  to the operating unit  24 , whereas the adder  23   b  in the adder  23  adds the numbers of fluorescent photons m 2   a  and m 2   b  to output the number of incoming fluorescent photons m 2  (=m 2   a +m 2   b ) at the second time gate ΔT 2  to the operating unit  24 . 
   The operating unit  24  solves Equation (8) for the received values of m 1  and m 2  to calculate the fluorescent lifetime τ: 
   
     
       
         
           
             
               
                 τ 
                 = 
                 
                   t 
                   
                     In 
                     ⁡ 
                     
                       ( 
                       
                         m 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         
                           1 
                           / 
                           m 
                         
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         2 
                       
                       ) 
                     
                   
                 
               
             
             
               
                 ( 
                 8 
                 ) 
               
             
           
         
       
     
   
   In the first embodiment, the half mirror  16  serves to separate the fluorescent photons to allow the accurate and sequential measurement of the fluorescent photons. With the correction of the numbers of photons measured by respective photodetectors  19   a  and  19   b  according to the Poisson distribution, which is the emission rate of the fluorescent photons, even when the count rate is increased from 0.01 as in the conventional technique to approximately 1, the calculation of the fluorescent lifetime is possible without the increase in errors. Then, the enhancement of the intensity of the exciting light pulse is allowed. Thus, with the half mirror  16  and the correction of the number of fluorescent photons, the count rate can be further increased to approximately as high as 2. In other words, the calculation of the fluorescent lifetime can be performed at nearly 200 times faster than the conventional technique. 
   In addition, though in the first embodiment the length of the first time gate ΔT 1  is set to be equal to the length of the second time gate ΔT 2 , they may not be equal. Still in addition, though in the first embodiment the time difference t of the start of the measurement is set to be smaller than the first time gate ΔT 1 , the time difference t may not be smaller as far as the time difference t is constant. 
   Next, a modification of the first embodiment will be described. In the first embodiment, one half mirror is arranged to separate the fluorescent photons to shorten the time for measuring the number of fluorescent photons to ½. In the modification, three half mirrors are arranged to further shorten the time for measuring the number of fluorescent photons to ¼. 
     FIG. 4  is a block diagram of a schematic structure of a scanning fluorescent microscope  200  as a modification of the first embodiment of the present invention. In  FIG. 4 , a measuring unit  13 A includes half mirrors  16   a  and  16   b  arranged at a subsequent stage of the half mirror  16 , and absorption filters  17   a   1 ,  17   a   2 ,  17   b   1 , and  17   b   2 , and photodetectors  19   a   1 ,  19   a   2 ,  19   b   1 , and  19   b   2 , arranged at a subsequent stage of the half mirrors  16   a  and  16   b . A controller  20 A includes signal processing units  21   e ,  21   f ,  21   g , and  21   h , correcting units  22   e ,  22   f ,  22   g , and  22   h , the adder  23   b  and the operating unit  24 . The same components with  FIG. 1  are denoted with the same reference characters. 
   The fluorescent photons coming into the half mirror  16  is split by the half mirror  16  and then by the half mirrors  16   a  and  16   b . Hence, the photodetectors  19   a   1 ,  19   a   2 ,  19   b   1 , and  19   b   2  output the electric signals Sa 1 , Sa 2 , Sb 1 , and Sb 2 , respectively, representing the four divided fluorescent photons. Signal processing units  21   e ,  21   f ,  21   g , and  21   h  receive the electric signals Sa 1 , Sa 2 , Sb 1 , and Sb 2 , respectively, to measure the number of fluorescent photons and output the measured number of fluorescent photons to the correcting units  22   e ,  22   f ,  22   g , and  22   h , respectively. The correcting units  22   e ,  22   f ,  22   g , and  22   h  correct the received numbers of fluorescent photons according to the Poisson distribution and supply the corrected numbers of fluorescent photons to the adder  23   b . The adder  23   b  adds the received numbers of fluorescent photons to output the result to the operating unit  24 . The operating unit  24  calculates the fluorescent lifetime based on the received numbers of fluorescent photons. 
   In the modification of the first embodiment, three half mirrors  16 ,  16   a , and  16   b  are arranged to split the fluorescent photons into four parts. Thus, the time required for the measurement of fluorescent photons can be made quarter the time required in the conventional technique. 
   In the modification of the first embodiment, the half mirrors  16 ,  16   a , and  16   b  are arranged to split the fluorescent photons into four parts, thereby shortening the measurement time of the number of fluorescent photons to quarter the conventional technique. However, more than three half mirrors may be arranged to further shorten the time required for measurement of the number of fluorescent photons. 
   Next, a second embodiment will be described. In the first embodiment, the half mirror  16  is arranged to shorten the time of measurement of the number of fluorescent photons. In the second embodiment, a polarization beam splitter is arranged to shorten the time for measurement of the number of fluorescent photons as well as to allow the measurement of the orientation of a fluorescent molecule in the sample. 
     FIG. 5  is a block diagram of a schematic structure of a scanning fluorescent microscope  300  according to the second embodiment. In  FIG. 5 , the scanning fluorescent microscope  300  includes a polarizer  26  arranged between the collimating lens  2  and the dichroic mirror  3  to enhance the linearity of the exciting light, and a polarization beam splitter  30  instead of the half mirror  16 . Further, analyzers  18   a  and  18   b  are arranged to supplant the limitation of polarizing characteristics of the polarization beam splitter  30 . Further, a controller  20 B includes a polarizing operating unit  27 . The same components with  FIG. 1  are denoted with the same reference characters. 
   As shown in  FIG. 5 , the exciting light pulse emitted from the laser source  1  is polarized by the polarizer  26  to a predetermined angle of polarization and directed to the sample  12 . When the fluorescent photon in the sample  12  has a transition moment, the sample would be excited only with the polarized light parallel to the transition moment and the fluorescence which is emitted during the transition from the excited state to the ground state is also parallel to the transition moment. Hence, when the polarization angle of the light polarized by the polarizer  26  is parallel with the transition moment of the fluorescent photon in the sample  12 , the polarized fluorescence is emitted from the sample  12 . 
   The polarized fluorescence emitted from the sample  12  moves the optical path of the exciting light pulse backwards from the objective lens  11  to the dichroic mirror  3  to enter a measuring unit  13 B. The fluorescence entering the measuring unit  13 B passes sequentially through the condenser lens  14  and the pinhole  15  to enter the polarization beam splitter  30 . The polarization beam splitter  30  polarizes the incident light by 90° (P-polarization) to generate transmissive light and polarizes the incident light by 0° (S-polarization) to generate reflective light. Hence, when the polarization angle of the incoming fluorescence into the polarization beam splitter  30  is 45°, the light is P-polarized with the probability of 50% and S-polarized with the probability of 50%. In other words, N fluorescent photons with 45° polarization angle are split in P-polarized portion and S-polarized portion each consisting of N/2 photons. 
   The S-polarized fluorescent photons enter a photodetector  19   c  via the absorption filter  17   a  and the analyzer  18   a , whereas the P-polarized fluorescent photons enter a photodetector  19   d  via the absorption filter  17   b  and the analyzer  18   b . The photodetector  19   c  outputs an electric signal Ss to the signal processing unit  21   a  for every incoming fluorescent photon, whereas the photodetector  19   d  outputs an electric signal Sp to the signal processing unit  21   b  for every incoming fluorescent photon. 
   This embodiment as shown in  FIG. 5  has the following advantageous functions. After the input of the electric signals Ss and Sp to the signal processing units  21   a  and  21   b , the correcting units  22   a  and  22   b  correct the counted number of fluorescent photons. To complete one function, for example, these corrected numbers of fluorescent photons are supplied sequentially to the adder  23  and the operating unit  24  and used to calculate the conventional fluorescent lifetime in a manner as mentioned above. And to complete an other function, each polarized corrected numbers of fluorescent photons are supplied to the polarizing operating unit  27  for the calculation of the numbers of fluorescent photons and/or lifetimes respectively. A lot of information such as the dynamics of a molecular movement, transition moment and a molecular orientation of a fluorescent molecule are obtained from these data sets. A display  25  selects and displays an image relating to the fluorescent lifetime and an image relating to the S-polarization and the P-polarization under the control of the controller  20 B. 
   In the second embodiment, with the arrangement of the polarizer  26  and the polarization beam splitter  30 , the fluorescent photons are split in two parts to shorten the time of measurement of the number of fluorescent photons to half. Further, with the display of the polarized fluorescent photons, the orientation of the fluorescent molecule in the sample  12  can be observed. 
   In the second embodiment, only one display  25  is provided to select and output the image of fluorescent lifetime distribution and the image relating to the polarization. However, two displays may be provided to constantly output and display two types of images. 
   Next, a modification of the second embodiment will be described. In the second embodiment, the polarization beam splitter  30  is arranged to shorten the time for measuring the number of fluorescent photons to half and to allow the observation of orientation of the fluorescent molecules. In the modification, however, a wavelength plate is arranged between the condenser lens  14  and the pinhole  15  to accommodate the change in the polarization angle of the fluorescence, so that the polarization angle of the fluorescence incident on the polarization beam splitter  30  can be adjusted. 
     FIG. 6  is a block diagram of a schematic structure of a scanning fluorescent microscope  400  according to the modification of the second embodiment. In  FIG. 6 , a measuring unit  13 C includes a ½ wavelength plate  29  as a polarization direction rotating unit between the condenser lens  14  and the pinhole  15 , and a controller  20 C includes a wavelength plate controller  28 . The wavelength plate controller  28  controls the rotation of the ½ wavelength plate  29 . The same components as in  FIG. 5  are denoted by the same reference characters. 
   The ½ wavelength plate  29  shifts the phase of the vertical polarization axis based on the difference in refractive indices of the horizontal polarization and the vertical polarization to change the angle of polarization of the incoming light. Though the polarization angle of the fluorescence emitted from the sample  12  is determined by the polarizer  26 , when the sample  12  is a living body, the polarization angle of the fluorescence might change. When the polarization angle of the fluorescence shifts from 45°, the probability of S-polarization and the probability of P-polarization at the polarization beam splitter  30  would not be the same, in other words, the fluorescent photons would not be equally split. To prevent such inconvenience, the polarization angle of the fluorescence is rotated by the wavelength plate  29  for adjustment so that the polarization angle of the incoming fluorescence to polarization beam splitter  30  is always maintained at 45°. The wavelength plate controller  28  receives the signals from the signal processing units  21   a  and  21   b  and controls the rotation angle of the ½ wavelength plate  29  so that the outputs from the photodetectors  19   c  and  19   d  are equal to each other. 
   In the modification, the ½ wavelength plate  29  serves to adjust the polarization angle of the incoming fluorescence into the polarization beam splitter  30  so that the change in the transition moment of the fluorescent molecule in the sample  12  can be accommodated. Thus, the fluorescent photons entering the polarization beam splitter  30  can be equally split and the accurate and high-speed measurement of the fluorescent lifetime is allowed. 
   Next, a third embodiment will be described. In the first embodiment, the half mirror  16  is arranged, and in the second embodiment the polarization beam splitter  30  is arranged to split the fluorescent photons to shorten the time of measurement of the number of fluorescent photons. In the third embodiment, however, a combination of the half mirror and the polarization beam splitter is arranged to shorten the time of measurement of the number of fluorescent photons as well as to allow the observation of the orientation of fluorescent molecule. 
     FIG. 7  is a block diagram of a schematic structure of a scanning fluorescent microscope  500  according to the third embodiment. In  FIG. 7 , the scanning fluorescent microscope  500  includes the polarizer  26  arranged between the collimating lens  2  and the dichroic mirror  3 , and polarization beam splitters  30   a  and  30   b  arranged at a subsequent stage of the half mirror  16 . Further, a controller  20 D includes a polarizing operating unit  27   b . The same components with  FIG. 4  are denoted with the same reference characters. 
   As shown in  FIG. 7 , the exciting light pulse emitted from the laser source  1  is polarized to a predetermined polarization angle by the polarizer  26  to be directed to the sample  12 . The sample  12  emits the polarized fluorescence which enters a measuring unit  13 D, sequentially passes through the condenser lens  14  and the pinhole  15 , and then enters the half mirror  16 . The half mirror  16  splits the incoming fluorescent photons regardless of the polarization angle leaving the polarization angles as they are. The split polarized fluorescent photons enter the polarization beam splitters  30   a  and  30   b  in a subsequent stage, respectively. The polarization beam splitters  30   a  and  30   b  split the incoming fluorescent photons in two parts depending on the polarization angle. Thus, the fluorescent photons entering the measuring unit  13 D are split eventually in four parts. 
   Four groups of fluorescent photons after splitting pass through absorption filters  17   e ,  17   f ,  17   g , and  17   h , analyzers  18   e ,  18   f ,  18   g , and  18   h , and photodetectors  19   e ,  19   f ,  19   g , and  19   h , respectively to be converted into electric signals. The electric signals are received and processed by the signal processing units  21   e ,  21   f ,  21   g , and  21   h , the correcting units  22   e ,  22   f ,  22   g , and  22   h , the adder  23   c , and the operating unit  24  for the calculation of the fluorescent lifetime. On the other hand, the corrected numbers of fluorescent photons output from the correcting units  22   e ,  22   f ,  22   g , and  22   h  are supplied into the polarizing operating unit  27   b  for the calculation of the numbers of fluorescent photons which are S-polarized and P-polarized. 
   In the third embodiment, with the arrangement of the half mirror  16  and the polarization beam splitters  30   a  and  30   b  in a subsequent stage of the half mirror  16 , the time for measuring the number of fluorescent photons is shortened to ¼. At the same time, the measurement of the orientation of fluorescent molecule in the sample  12  is allowed. 
   In the third embodiment, one half mirror  16  is arranged and two polarization beam splitters  30   a  and  30   b  are arranged in a subsequent stage of the half mirror  16 . However, more than one half mirrors may be arranged and the polarization beam splitter may be arranged at the last stage. 
   Further, a wavelength plate may be arranged between the condenser lens  14  and the pinhole  15  to accommodate the change in the polarization angle of fluorescence so that the polarization angle of the incoming fluorescence to the polarization beam splitters  30   a  and  30   b  is constant. 
   Still further, though in the second and the third embodiments, the polarizer  26  is employed to enhance the linearity of polarization of the exciting light pulse, the use of polarizer  26  may not be necessary when the exciting light source provides highly linearly polarized laser beam. 
   As described above, the scanning fluorescent microscope according to any one of the embodiments has advantages of calculating the fluorescent lifetime with little error in a short time by using a splitter guiding the fluorescent photons to several optical paths. 
   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.