Abstract:
There is provided a light measurement apparatus includes a light source, a container which includes a sample tank, a lens which condenses light from a sample accommodated in the sample tank or from the container which are exited by a exciting light from the light source, a photodetector which detects light from the sample accommodated in the sample tank or from the container, wherein the light passes through the lens, a position detecting unit which detects a position of the sample tank based on a detection output from the photodetector, and a position adjusting unit which sets the container or the lens based on a detection output from the position detecting unit.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This is a Continuation Application of PCT Application No. PCT/JP2005/004566, filed Mar. 15, 2005, which was published under PCT Article 21(2) in Japanese.  
         [0002]     This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2004-076342, filed Mar. 17, 2004, the entire contents of which are incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION  
       [0003]     1. Field of the Invention  
         [0004]     The present invention relates to a light measurement apparatus and a light measurement method which mark a sample accommodated in a microplate with a fluorescent material, and irradiate this sample with light to perform fluorescence correlation spectroscopy (FCS) of an intensity fluctuation of fluorescence emitted by the sample or correlation analysis of an intensity fluctuation of fluorescence or an intensity fluctuation of scattered light emitted by a microscopic substance such as a carrier particle.  
         [0005]     2. Description of the Related Art  
         [0006]     In recent years, a configuration, an interaction or the like of a cell of an organism or a protein material is examined by using light. For example, a sample such as a protein material is directly marked with fluorescence, insides of a plurality of wells as circular grooves of a microplate accommodating the sample are irradiated with light such as a laser beam to excite a fluorescent material, and an intensity fluctuation of the fluorescence is detected, thereby examining a reaction or a change in conformation of the sample. According to this measurement method using light, a coupling reaction of a protein material such as signal transmission which occurs in or out of a living cell can be accurately measured by highly sensitive fluorescence detection.  
         [0007]     Meanwhile, a microplate is extensively used in such measurement. That is because using the microplate can measure many samples at a time with a small sample amount of the level of several tens of micro-liters. Although the usually utilized microplate has a size of 127 mm×85 mm and 96 wells, a microplate having many wells is commonly used as a recent tendency in order to save a sample amount or realize a high throughput. However, when many wells are arranged in the microplate, a size per well is reduced. A diameter of one well is approximately 4.5 mm in a microplate having 384 wells, whereas a diameter is as small as approximately 1.5 mm in a microplate having 1,536 holes.  
         [0008]     When such a microplate provided with many very small wells is held on a sample stage and a light irradiation position is adjusted by a regular positioning method such as visual confirmation, using an objective lens with a high magnifying power as a condenser lens makes it difficult to accurately set a light sight on a measurement well, resulting in a risk of displacement. In order to accurately manage measurement data for each well, positional data must be matched with a measurement region, and hence wells of the microplate must be positioned with extreme accuracy.  
         [0009]     As prior art of positioning a microplate, the following two methods have been proposed.  
         [0010]     According to a method disclosed in Jpn. Pat. Appln. KOKAI Publication No. 2002-14035, some of wells are determined as “position recognizing wells”, and a fluorescent material is injected into these wells so that a position of each well can be visually confirmed. Further, an entire region of a microplate is illuminated with light, fluorescence emitted by the fluorescent material included in a sample in each well is collectively captured by using a CCD camera, and an intensity of fluorescence emitted by the sample in each well is measured with respect to all the wells.  
         [0011]     Furthermore, according to a method disclosed in specification of U.S. Pat. No. 6,258,326, a microplate itself is directly processed, a plurality of reference grooves are arranged, and a coordinate of each reference groove is optically measured to position the microplate. In particular, when the number of wells is as large as 1,536, the plurality of reference grooves are arranged between the wells to further increase positioning accuracy.  
       BRIEF SUMMARY OF THE INVENTION  
       [0012]     According to one aspect of the present invention, there is provided a light measurement apparatus comprising: a light source; a container which includes a sample tank; a lens which condenses light from a sample accommodated in the sample tank or from the container which are exited by a exciting light from the light source; a photodetector which detects light from the sample accommodated in the sample tank or from the container, wherein the light passes through the lens; a position detecting unit which detects a position of the sample tank based on a detection output from the photodetector; and a position adjusting unit which sets the container or the lens based on a detection output from the position detecting unit.  
         [0013]     Further, according to one aspect of the present invention, there is provided a light measurement method comprising: holding a container which includes a sample tank; condensing light from the container or from a sample accommodated in the sample tank by a lens; detecting the light from the container or from the sample by a photodetector, wherein the light passes though the lens; and moving a position of the lens or a position of the sample tank based on a detection output from the photodetector in such a manner that an optical axis of the lens runs through the sample tank.  
         [0014]     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  
       [0015]     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.  
         [0016]      FIG. 1  is a view showing a configuration of a light measurement apparatus according to a first embodiment of the present invention.  
         [0017]      FIG. 2  is an enlarged schematic view showing a microplate and an objective lens.  
         [0018]      FIG. 3  is a view showing an operation of obtaining a central point of each well.  
         [0019]      FIG. 4  is a view showing an operation of obtaining a central point of a well.  
         [0020]      FIG. 5A  is a view showing a processing method of a detection signal.  
         [0021]      FIG. 5B  is a view showing a processing method of a detection signal.  
         [0022]      FIG. 6A  is a view showing an operation of obtaining a central point of a well.  
         [0023]      FIG. 6B  is a view showing the operation of obtaining the central point of the well.  
         [0024]      FIG. 6C  is a view showing the operation of obtaining the central point of the well.  
         [0025]      FIG. 6D  is a view showing the operation of obtaining the central point of the well.  
         [0026]      FIG. 6E  is a view showing the operation of obtaining the central point of the well.  
         [0027]      FIG. 7  is a view illustrating a method of obtaining a coordinate of a central position.  
         [0028]      FIG. 8  is a view showing a configuration of a light measurement apparatus according to a third embodiment of the present invention.  
         [0029]      FIG. 9  is a view showing a configuration of a light measurement apparatus according to a fourth embodiment of the present invention.  
         [0030]      FIG. 10A  is a view showing a configuration of a microplate and wells.  
         [0031]      FIG. 10B  is a view showing a configuration of a microplate and wells.  
         [0032]      FIG. 11A  is a view showing an operation of obtaining a central point of a well.  
         [0033]      FIG. 11B  is a view showing the operation of obtaining the central point of the well.  
         [0034]      FIG. 11C  is a view showing the operation of obtaining the central point of the well.  
         [0035]      FIG. 11D  is a view showing the operation of obtaining the central point of the well.  
         [0036]      FIG. 11E  is a view showing the operation of obtaining the central point of the well. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
     First Embodiment  
       [0037]      FIG. 1  is a view showing a configuration of a light measurement apparatus according to a first embodiment of the present invention. A basic apparatus configuration of the light measurement apparatus according to this embodiment is based on a confocal optical microscope. A configuration and operation of the light measurement apparatus will now be described with reference to  FIG. 1 .  
         [0038]     The light measurement apparatus according to this embodiment is provided with two types of light sources  1   a  and  1   b . A helium-neon laser (oscillation output: 2 mW, wavelength: 633 nm) is used for the light source  1   a , and an argon laser (oscillation output: 10 mW, wavelength: 488 nm) is used for the light source  1   b . Traveling directions of laser beams emitted by the light source  1   a  and the light source  1   b  are changed by a mirror, and then light paths of the two laser beams are combined into one light path by a dichroic mirror  2 . The combined light beam is converted into a collimated light beam whose beam diameter has been increased by a lens  3 , travels, is reflected by a dichroic mirror  4 , and reaches an objective lens  5 .  
         [0039]     Light condensed by the objective lens  5  is applied to the inside of each well  8  in a microplate  7  mounted and fixed on a sample stage  6 . A sample stage XY axis driving mechanism  18  drives the sample stage  6  to adjust a horizontal position of the microplate  7  and an objective lens Z axis adjusting mechanism  17  drives the objective lens  5  to adjust a vertical position of the microplate  7  in such a manner that a sample in each well  8  is irradiated with a region (which will be referred to as a confocal region) condensed by the objective lens  5 . It is to be noted that a plane on which the microplate  7  is driven by the sample stage  6  is called an XY plane, and an axis along which the objective lens  5  is driven by the objective lens Z axis adjusting mechanism  17  is called a Z axis. The Z axis is an axis vertical to the XY plane.  
         [0040]     The microplate  7  is formed of a generally used resin and glass as materials. As shown in  FIG. 1 , many wells  8  are arrange in the microplate  7 . Although not shown, a bottom surface of each well  8  is a window formed of a material such as glass through which visible light is transmitted.  
         [0041]      FIG. 2  is an enlarged schematic view showing the microplate  7  and the objective lens  5  arranged below the microplate  7 . The objective lens  5  is configured to face the bottom surface of each well  8 .  
         [0042]     As the objective lens  5 , for example, a ×40 immersion objective lens (NA0.9) is used, and a space between the bottom surface of the microplate  7  and an end of the objective lens  5  is filled with immersion water. In this embodiment, a focal position of a laser beam is a central part of the well  8  in the horizontal direction (X-Y axis), and a position which is 100 μm above an upper wall position of the bottom surface of the well  8  in the vertical direction (Z axis). Further, as to a size and a shape of a confocal region in the well  8 , the well  8  has a substantially cylindrical shape having a diameter of approximately 0.6 μm and a length of approximately 2 μm.  
         [0043]     The laser beam condensed by the objective lens  5  excites fluorescent molecules in the sample, and fluorescence is generated by the fluorescent molecules. The generated fluorescence is again transmitted through the objective lens  5  and the dichroic mirror  4 , and enters a barrier filter  9 .  
         [0044]     The dichroic mirror  4  is manufactured by applying multiplayer film coating on one side of a flat glass sheet so that reflection spectral characteristics become optimum. It is to be noted that, as the dichroic mirror  4 , not only a planar mirror but also a prism-shaped mirror can be used.  
         [0045]     The barrier filter  9  is formed into a discotic shape and has permeation characteristics matched with an emission spectrum of the fluorescence. That is, it transmits light in a wavelength band of the fluorescence as signal light alone therethrough. As a result, it is possible to block off scattered light generated in the sample container or noise light which is reflected from a wall or the like of the well  8  and returns to an incident light path. That is because the noise light as background light is blocked off since its wavelength is different from that of the fluorescence.  
         [0046]     The signal light transmitted through the barrier filter  9  is transmitted through a lens  10  to be converted into condensed light, reflected by a mirror  11 , and condensed on a pinhole surface of a pinhole  12  arranged behind the lens  10 . That is, an opening surface of the pinhole  12  matches with a focal plane of the lens  10 . A diameter of the pinhole  12  is variable. The background light can be removed from a region other than a light confocal region formed in the well  8  by the pinhole  12 .  
         [0047]     A photodetector  13  is arranged in the vicinity of a rear position of the pinhole  12 . The signal light received by the photodetector  13  is weak light and has a photon pulse. Therefore, as the photodetector  13 , for example, an avalanche photodiode (APD) or a weak photodetector such as a photomultiplier is used. The photodetector  13  converts the signal light into a photocurrent pulse which is an electric signal. The converted electric signal enters a signal processor  14  to be amplified and waveform-shaped, and it is converted into an on/off voltage pulse to be led to a computer  15 .  
         [0048]     This voltage pulse signal is stored as data in a memory (not shown) in the computer  15 , and then an arithmetic operation such as correlation analysis is carried out based on this data. Furthermore, analysis results such as an intensity of the fluorescence, a life duration of the fluorescence, an auto-correlation function of an intensity fluctuation of the obtained fluorescence or a cross-correlation function are presented on a screen of the computer  15 .  
         [0049]     Moreover, the computer  15  controls each portion of the light measurement apparatus to measure a central coordinate of the bottom surface of the well  8  in the microplate  7 , operates the sample stage XY axis driving mechanism  18  based on the measured value, and then moves the microplate  7  in such a manner that a central position of the bottom surface of the well  8  is irradiated with light emitted by the light sources.  
         [0050]     It is to be noted that Rhodamine Green (RhG) and Cy5 are used as fluorescent materials in this embodiment.  
         [0051]     Rhodamine Green has a peak wavelength of absorption in the vicinity of 490 nm and a peak of an emission wavelength in the vicinity of 530 nm. Therefore, Rhodamine Green is excited by the argon laser. Additionally, Cy5 has a peak wavelength of absorption in the vicinity of 640 nm and a peak of an emission wavelength in the vicinity of 670 nm. Therefore, Cy5 is excited by the helium-neon laser.  
         [0052]     A description will now be given as to a method of positioning a central position of the well  8  in the microplate  7  according to the first embodiment.  
         [0053]     Procedure 1: a well  8  as a reference is first determined in the microplate  7 . For example, a well  8  at an uppermost left corner of the microplate  7  shown in  FIG. 3  is determined as a reference well  8 , and a central coordinate of this well  8  is determined as (x 1 , y 1 ).  
         [0054]     Procedure 2: the held sample stage  6  is first moved in the XY plane by operating the sample stage XY axis driving mechanism  18 , and a spot of a laser beam is arranged on a wall upper surface which is the outside of the reference well  8  at the uppermost left corner. At this time, as shown in  FIG. 4 , the spot of the laser beam is a position P in the vicinity of an upper end of the well  8 .  
         [0055]     Procedure 3: then, as shown in  FIG. 4 , the sample stage is moved on a trajectory R 1  along a y axis, and a count rate as an intensity of light generated by the well  8  in the microplate  7  at this time is read.  
         [0056]     A detection signal output by the photodetector  13  is a signal in which noise is superimposed on a count rate signal which is an intensity of light as shown in  FIG. 5A . A threshold value is determined with respect to this detection signal, and waveform shaping is carried out to convert the detection signal into an on/off digital signal shown in  FIG. 5B .  
         [0057]     Procedure 4: a y coordinate at the center of a rectangular shape of the obtained on/off digital signal is acquired by a calculation.  
         [0058]     Procedure 5: subsequently, as shown in  FIG. 4 , the sample stage is moved on a trajectory R 2  along an x axis, and a count rate as an intensity of light generated by the well  8  in the microplate  7  at this time is read. Additionally, the signal processing described in Procedure 3 is carried out to obtain an on/off digital signal.  
         [0059]     Procedure 6: an x coordinate at the center of a rectangular shape of the obtained on/off digital signal is acquired by a calculation.  
         [0060]     If the coordinate of the central point of one well  8  is obtained by the above-described procedures, moving the sample stage  6  can acquire a central point of each well  8 .  
         [0061]     For example, as shown in  FIG. 3 , a y coordinate of the central point is fixed, the sample stage  6  is moved along the x axis to record a detection output with time, thereby detecting an x coordinate of a boundary wall surface of the well  8 . It is assumed that X 1  is an x coordinate of a first signal obtained from a wall surface of the well  8 , and X 2  is an x coordinate of the next signal obtained from the wall surface of the well  8 . Then, X coordinates are sequentially determined. Further, an x coordinate (x n ) of a central point of an nth well  8  can be obtained based on the following Expression (1). 
 
 x   n   =X   2n-1 +( X   2n   −X   2n-1 )/2  Expression (1)
 
         [0062]     X 1 : X coordinate at a left end of a wall surface of a first well  8   
         [0063]     X 2 : X coordinate at a right end of the wall surface of the first well  8   
         [0064]     X 2n-1 : X coordinate at a left end of a wall surface of an nth well  8   
         [0065]     X 2n : X coordinate at a right end of the wall surface of the nth well  8   
         [0066]     Likewise, an x coordinate at a central point is fixed, and the sample stage  6  is moved along the y axis, thereby obtaining a y coordinate (y n ) at the central point of the nth well  8  based on the following Expression (2) 
 
 y   n   =Y   2n-1 +( Y   2n   −Y   2n-1 )/2  Expression (2)
 
         [0067]     Y 1 : Y coordinate at the left end of the wall surface of the first well  8   
         [0068]     Y 2 : Y coordinate at the right end of the wall surface of the first well  8   
         [0069]     . . .  
         [0070]     Y 2n-1 : Y coordinate at the left end of the wall surface of the nth well  8   
         [0071]     Y 2n : Y coordinate at the right end of the wall surface of the nth well  8   
         [0072]     Moving the sample stage  6  in this manner can obtain the central point of the bottom surface of each of all the wells  8  in the microplate  7 . Moreover, when measuring each well  8 , accurate positioning can be performed by moving the sample stage  6  based on the obtained central position information of each well  8  in such a manner that a spot of the laser beam is placed at the center of the bottom surface of each well  8 .  
       Second Embodiment  
       [0073]     Although an apparatus configuration of a light measurement apparatus according to a second embodiment of the present invention is the same as that according to the first embodiment, procedures of obtaining a central coordinate of each well  8  are different from those in the first embodiment. Therefore, like reference numerals denote parts equal to those in the first embodiment, thereby eliminating the detailed explanation.  
         [0074]     A description will now be given as to a method of positioning a central position of each well  8  in a microplate  7  according to the second embodiment.  
         [0075]     Procedure 1: a well  8  as a reference in the microplate  7  is determined in advance. For example, a well  8  at an uppermost left corner of the microplate  7  shown in  FIG. 3  is determined as a reference well  8 , and a central coordinate of this well  8  is determined as (x 1 , y 1 ).  
         [0076]     Procedure 2: a held sample stage  6  is moved in an XY plane by operating a sample stage XY axis driving mechanism  8  to arrange a spot of a laser beam on a wall upper surface which is the outside of the reference well  8  at the uppermost left corner. At this time, the spot of the laser beam is a position P in the vicinity of an upper end of the well  8  as shown in  FIG. 6A .  
         [0077]     Procedure 3: then, the sample stage  6  is moved along an x axis as shown in  FIG. 6A . At this time, a count rate which is an intensity of light generated by the well  8  in the microplate  7  is read.  
         [0078]     A detection signal output by a photodetector  13  is a signal in which noise is superimposed on a count rate signal which is an intensity of light as shown in  FIG. 5A . A threshold value is set with respect to this detection signal, waveform shaping is performed, and this detection signal is converted into an on/off digital signal shown in  FIG. 5B , thereby obtaining a detection signal.  
         [0079]     Procedure 4: the sample stage  6  is moved along a y axis by a predetermined distance to change a y coordinate, and Procedure 3 is executed to obtain a detection signal  1 .  
         [0080]     Procedure 5: as shown in  FIGS. 6B  to  6 E, Procedures 3 and 4 are repeatedly executed to obtain detection signals  2  to  5 .  
         [0081]     Procedure 6: the detection signals  1  to  5  are compared, and a value of the y axis which outputs a detection signal  3  having the largest width of a rectangular shape is a y coordinate (y 1 ) which gives the central position of the well  8 .  
         [0082]     Procedure 7: Procedures 2 to 6 are repeated while moving the sample stage  6  along the y axis. Moreover, an x coordinate (x 1 ) which gives the central position of the well  8  is obtained.  
         [0083]     After the coordinate of the central point of one well  8  is obtained in this manner, moving the sample stage  6  can acquire a central point of each of all the wells  8  in the microplate  7  like the first embodiment.  
       Variation of Second Embodiment  
       [0084]     The method of detecting x and y coordinates at a maximum width position on a boundary wall surface of each well  8  is not restricted to the method shown in  FIGS. 6A  to  6 E. For example, after the detection signal  1  is obtained, the sample stage  6  is moved along the y axis by a fixed moving distance H, and the sample stage  6  is moved from this position along the x axis, thereby obtaining the detection signal  5 . Then, a coordinate at a central position O is obtained based on a geometric relationship depicted in  FIG. 7 , and this may be determined as the y coordinate at the maximum width position on the boundary wall surface of the well  8 .  
         [0085]     Incidentally, since the respective wells  8  basically have the same diameter and they are arranged on the microplate  7  at equal intervals, coordinates at respective central points of a plurality of representative wells  8 , e.g., wells  8  at four corners may be obtained and coordinates at central points of other wells  8  may be acquired by a calculation without obtaining coordinates at central points of all the wells  8  on the microplate  7 .  
         [0086]     Alternatively, coordinates at central points of wells  8  in one lateral column and one vertical column at endmost positions may be obtained, then coordinates at central points of all the wells  8  may be acquired by a calculation, and positioning may be executed by using the sample stage  6 . Even if the microplate  7  is obliquely inclined, since obtaining coordinates at central points of at least two wells  8  can calculate a degree of inclination, positions of wells  8  can be determined considering this inclination.  
         [0087]     It is to be noted that the above-described measurement of the central point of each well  8  and driving control over the sample stage  6  based on this measured value can be realized when the computer  15  controls the respective portions in the light measurement apparatus in cooperation.  
         [0088]     That is, the computer  15  controls all of the respective portions in the light measurement apparatus to measure a central coordinate of a bottom surface of each well  8  in the microplate  7 , operates the sample stage XY axis driving mechanism  18  based on this measured value, and then moves the microplate  7  in such a manner that a central position of the bottom surface of this well  8  is irradiated with light emitted by the light sources.  
       Third Embodiment  
       [0089]      FIG. 8  is a view showing a configuration of a light measurement apparatus according to a third embodiment of the present invention. A basic configuration of the third embodiment is the same as the configuration of the first embodiment shown in  FIG. 1  except that light sources are not required, and hence like reference numerals denote like parts, thereby eliminating the detailed explanation.  
         [0090]     An objective lens plane moving mechanism  19  is connected with an objective lens  5 , and driving by a stepping motor is controlled by a computer (not shown) to match a position of each well  8  with an optical axis of the objective lens  5 . An adjustment range of the objective lens plane moving mechanism  19  is as small as several tens of μm or below, and the adjustment is carried out as a final adjustment of a well position. Here, the objective lens plane moving mechanism  19  may be driven by using not only the stepping motor but also an ultrasonic motor.  
         [0091]     In this embodiment, fluorescence generated by a resin of a microplate main body is determined as a measurement signal. As shown in  FIG. 10A , a wall of each well  8  in the microplate  7  is formed of a resin, and a bottom surface of the same has a configuration obtained by bonding a glass sheet. A resin portion of a microplate main body is formed of a mold member obtained by molding. As a material, it is possible to use polycarbonate, polystyrene, acrylic and others. When such a resin is used for at least a wall portion of the microplate main body, fluorescence is generated by the resin portion. However, in this case, an optically transparent and nonluminescent glass sheet is used for each well portion, and it is bonded to the resin portion of the microplate main body. It is to be noted that the microplate  7  may be manufactured by fitting the resin portion of the microplate main body to the nonluminescent glass sheet constituting the bottom surface as shown in  FIG. 10B .  
         [0092]     Measurement procedures will now be described with reference to  FIGS. 11A  to  11 E. First, the microplate  7  is set on the sample stage  6  in a state where a sample is not accommodated in each well. The sample stage  6  is moved by a small distance along the y axis. At this time, a point P which is a laser spot describes a trajectory indicated by an arrow. At this time, fluorescence cannot be basically obtained from each well portion, but fluorescence is generated by at least a wall portion of the microplate main body. Based on this detection signal, like the second embodiment shown in  FIGS. 6A  to  6 E, a central position of each well  8  is determined. It is good enough to perform the same operation with respect to the x axis direction as that executed in regard to the y axis direction.  
         [0093]     Then, a power supply of the objective lens moving mechanism  19  is turned on, an optimum position of the objective lens on the xy plate is determined while detecting a fluorescence signal from the wall portion, and the objective lens  5  is moved by a necessary distance. After determining the central position of each well  8 , the sample is injected into the well  8 , and light emission from the sample is detected. In this embodiment, since the fluorescence generated by the resin of the microplate main body is determined as a measurement signal, the sample may be or may not be accommodated in each well.  
         [0094]     When the sample is accommodated in each well and a light intensity of light emission from the sample is large, an output signal from the photodetector becomes larger than the fluorescence generated by the resin of the microplate main body when the well portion faces the objective lens. Therefore, on/off states of detection outputs from the photodetector are reversed as compared with detection signals shown in  FIGS. 11A  to  11 E. On the other hand, when the sample is accommodated in each well and a light intensity of the fluorescence generated by the sample is smaller than that of the fluorescence generated by the resin of the microplate main body, output signals from the photodetector when each well portion faces the objective lens become equal to those shown in  FIGS. 11A  to  11 E.  
         [0095]     The apparatus shown in  FIG. 8  is likewise used when measuring a luminous phenomenon such as chemiluminescence or bioluminescence. A well position determining method is substantially the same as that of the second embodiment shown in  FIGS. 6A  to  6 E, and light emission caused due to the sample from the well  8  is detected and determined.  
         [0096]     A description will now be given as to an example where the apparatus shown in  FIG. 8  is used to perform enzymatic immunoassay of, e.g., alpha-fetoprotein (AFP) which is a major cancer marker. First, an anti-AFP antibody is sensitized to glass particles having a diameter of approximately 1 to 10 μm, marked with oxygen alkaline phosphatase, suspended in a buffer fluid, and accommodated in each well. A specimen material such as blood is added to cause an antigen antibody reaction at a room temperature. Furthermore, glass particles which have not contributed to the reaction are removed by cleaning, and a chemiluminescent substrate AMPPD (2-dioxetane disodium salt) is added to the remaining solution. At this time, AMPPD reacts with oxygen alkaline phosphatase, and chemiluminescence thereby occurs. A luminescence intensity of this chemiluminescence is measured by the photodetector, thus determining an AFP concentration in the specimen material.  
       Fourth Embodiment  
       [0097]      FIG. 9  is a view showing a configuration of a light measurement apparatus according to a fourth embodiment of the present invention. In the fourth embodiment, a resin portion of a microplate main body is irradiated with a laser beam, and fluorescence generated by this resin portion is measured to detect a position of each well. Since a basic configuration of the fourth embodiment is the same as that of the first embodiment except that the number of light source is one, like reference numerals denote like parts, thereby eliminating the detailed explanation.  
         [0098]     As a light source, a helium-cadmium laser having an output of 50 mW and a wavelength of 325 nm is used. Although an argon laser having a wavelength of 488 nm or a helium-neon laser having a wavelength of 633 nm may be used as the light source like the first embodiment, using the helium-cadmium laser having a wavelength of 325 nm can obtain a further high fluorescence intensity since its wavelength is close to an absorption wavelength of a resin constituting the microplate main body.  
         [0099]     After light emitted by the light source  1   c  is reflected by mirrors twice, the light is led to a lens  3  to provide collimated light. This collimated light is reflected by a dichroic mirror  4  to be led to an objective lens  5 , and condensed onto a microplate  7 .  
         [0100]     Measurement procedures will now be described with reference to  FIGS. 11A  to  11 E. First, the microplate  7  is set on a sample stage  6  in a state where a sample is not accommodated in each well  8 . The microplate main body is irradiated with the light from the light source  1   c  through the objective lens  5 . The sample stage  6  is moved by a small distance along a y axis. At this time, a point R which is a laser spot describes a trajectory indicated by an arrow. At this time, fluorescence cannot be basically obtained from each well portion, but the fluorescence is generated by at least a wall portion of the microplate main body. A central position of each well  8  is determined based on this detection signal like the second embodiment shown in  FIGS. 6A  to  6 E. It is good enough to perform the same operation with respect to an x axis direction as that executed in the y axis direction.  
         [0101]     Then, a power supply of an objective lens moving mechanism  19  is turned on, and an optimum position of the objective lens  5  on an xy plane is determined to move the objective lens  5  by a necessary distance. After determining an optimum position of the well  8 , a sample is injected into the well  8  to detect light emission from the sample. In this embodiment, in order to determine fluorescence generated by the resin of the microplate main body as a measurement signal, the sample may be or may not be accommodated in each well.  
         [0102]     It is to be noted that the description has been given as to the microplate  7  having the round wells  8  in this embodiment, but the present invention is not restricted thereto, and a central position of each well  8  can be determined by the same operation in the microplate  7  having, e.g., rectangular wells  8 . Furthermore, the number of wells is not restricted to  96 , and  384  wells or the like can be used.  
         [0103]     Moreover, measurement can be likewise performed with respect to chemiluminescence or bioluminescence.  
       Effects of Embodiment  
       [0104]     In the foregoing embodiment, the commonly available microplate  7  is used as it is, a position of each well  8  in the microplate  7  is optically measured, a central position of the same on the XY plane is obtained, and a light spot position is accurately positioned in order to measure the sample. Additionally, at this time, optical means for measuring a position of each well  8  in the microplate  7  is not specially arranged, but the optical system which measures the sample is used for position measurement as it is, and hence the structure is simple.  
         [0105]     Further, although the microplate  7  is used to calculate a reference position to accurately grasp a position of each well  8 , the well  8  may be a well  8  in which the sample is accommodated or a well  8  in which a commonly used fluorescence material is accommodated. Therefore, a special chemical is not required, and the wells  8  in the microplate  7  are not wastefully used.  
         [0106]     Furthermore, although determining one reference point can determine a coordinate at a central point of each of other wells  8  in usual measurement, determining at least two reference points by the above-described method can calculate a degree of inclination even if the microplate  7  is obliquely inclined, thus obtaining coordinates at central points of all the wells  8 .  
         [0107]     Furthermore, since the configuration of the optical system which is used for actual measurement of the sample is used as it is, a special mechanism or device for positional detection does not have to be provided.  
         [0108]     Moreover, even in case of the microplate  7  such as a microplate  7  having 1,536 wells in which a size per well is extremely small, since accurate positioning and measurement can be carried out, measurement data for each well  8  can be accurately managed.  
         [0109]     It is to be noted that the microplate  7  is used for calculation of a reference point in order to accurately grasp a position of each well  8 , but the method described in this embodiment can be applied to the well  8  in which the sample is not accommodated. In this case, reflected light rather than fluorescence is returned from the microplate  7 . Therefore, using a beam splitter in place of the dichroic mirror  4  can suffice.  
         [0110]     It is to be noted that the functions described in each foregoing embodiment can be realized by not only using hardware but also reading a program in which each function is written by software into a computer. Additionally, each function may be configured by appropriately selecting software or hardware.  
         [0111]     Further, each function can be also realized by reading a program stored in a non-illustrated storage medium into a computer. Here, the storage medium in this embodiment may take any conformation as long as it is a storage medium which can store a program and can be read by a computer.  
         [0112]     The present invention is not limited directly to the above embodiments but may be practiced with constitutional elements thereof modified without deviating from the subject matter of the invention in practical phases. Further, various inventions may be formed by appropriately combining plural constitutional elements disclosed in any of the above embodiments. For example, several constitutional elements may be removed from all constitutional elements suggested in any of the embodiments. Furthermore, constitutional elements may be combined between different embodiments.