Abstract:
A fluorescent detection apparatus relates to an analysis technique for qualitatively detecting or quantifying biomolecules by producing an evanescent field on a surface of a substrate, exciting fluorescently labelled biomolecules on the substrate surface in the evanescent field, and detecting the resultant fluorescent light emitted from the biomolecules. The fluorescent detection apparatus has a configuration in which a well is provided in a surface opposing to a sample substrate of a prism, the well is filled with a matching liquid, and the matching liquid is filled between the sample substrate and the prism, thereby improving operability and providing a stable evanescent field.

Description:
INCORPORATION BY REFERENCE 
       [0001]    The present application claims priority from Japanese application JP2007-335612 filed on Dec. 27, 2007, the content of which is hereby incorporated by reference into this application. 
       FIELD OF THE INVENTION 
       [0002]    The present invention relates to an analysis technique for qualitatively detecting or quantifying biomolecules by producing an evanescent field on a surface of a transparent substrate, exciting fluorescently labelled biomolecules in a liquid sample supplied on the substrate surface in the evanescent field, and detecting the resultant fluorescent light emitted from the biomolecules. 
       DESCRIPTION OF RELATED ART 
       [0003]    In the related art, single fluorescent molecule has been observed using an evanescent field produced on a substrate surface by irradiating excitation light emitted from an light source to a transparent sample substrate and totally reflecting the excitation light on the surface of the sample substrate. 
         [0004]    For example, as disclosed in “Funatsu et al., Nature Vol. 374, 555-559 (1995)”, for single molecule fluorescence detection, in order to produce an evanescent field, a prism plane and a sample substrate are oppositely arranged in parallel to each other and a matching liquid for matching a refractive index of the prism plane and a refractive index of the sample substrate is filled between the prism plane and the sample substrate. 
         [0005]    In addition, as disclosed in “Braslavsky et al., PNAS Vol. 100, 3960-3964 (2003)”, sequencing single DNA molecule using a total-internal-reflection fluorescence microscopy is conducted. Here, lasers having wavelengths of 532 nm and 635 nm are used for fluorescence detection of a fluorophore Cy3 and a fluorophore Cy5, respectively. When single target DNA molecules are immobilized on a sample substrate filled with a solution using a biotin-avidin protein binding and a primer labelled with Cy3 is introduced into the solution such that the primer has constant concentration by a solution exchange, single fluorescence-marked primer molecules are hybridized with the target DNA molecules. At this time, since Cy3 exists in an evanescent field, the existence of the target DNA molecules hybridized with Cy3-labelled primer can be recognized by the fluorescence detection. After the fluorescence detection of Cy3, Cy3 is photobleached by irradiating Cy3 with excitation light of high power of 532 nm, thereby suppressing later fluorescent light emission. Next, when polymerase and dNTP (N being one of A, C, G and T), which is a kind of base labelled with Cy5 mono-molecule, are introduced in the solution such that the polymerase and the dNTP have constant concentration, respectively, by solution exchange, fluorescently labelled dNTP molecules are incorporated into elongation end of primer molecules only when the fluorescently labelled dNTP molecules have complementary relation with target DNA molecules. At this time, since Cy5 exists in an evanescent field, the incorporation can be checked at a position of the target DNA molecules by fluorescence detection. After the check, Cy5 is photobleached by irradiating Cy5 with excitation light of high power of 635 nm, thereby suppressing later fluorescent light emission. By repeating the above-described dNTP incorporating reaction process sequentially and cyclically for kinds of bases, for example, A→C→G→T→A→ (cyclic polymerase reaction), it is possible to determine a sequence having a complementary relation with the target DNA molecules. In addition, by immobilizing a plurality of target DNA molecules within the same field of a fluorescence detection image and performing the dNTP incorporating reaction process in parallel, a simultaneous DNA sequencing of the plurality of target DNA molecules becomes possible. It is expected that the number of simultaneous parallel processes at this time can rapidly increase in comparison with a conventional electrophoresis-based DNA sequencing. 
         [0006]    In such a conventional single molecule fluorescence detection, by flowing a matching liquid onto a prism when a sample substrate is arranged, the matching liquid is filled between the sample substrate and the prism such that air is not introduced between the sample substrate and the prism. In this case, however, if the amount of the flown matching liquid is excessive, an apparatus may become dirty due to matching liquid leaked out during replacement of the sample substrate or the like, which may have an adverse effect on stage precision or measurement. 
         [0007]    For the purpose of overcoming this problem, JP-A-8-136554 discloses a method of receiving matching liquid which overflows in replacement of a sample substrate by providing a grooved oil puddle in the outer circumference of a prism. 
         [0008]    In the conventional single molecule fluorescence detection, when the sample substrate is arranged, it is required to fill the matching liquid between the prism and the sample substrate in such a manner that air is not introduced therebetween. In this case, if the amount of flown matching liquid is small, it becomes difficult for the sample substrate to move due to surface energy of the matching liquid. Even if the sample substrate does move, the matching liquid moves with horizontal movement of the sample substrate and air is introduced between the sample substrate and the prism, thereby preventing excitation light from arriving at the sample substrate. On the other hand, if the amount of flown matching liquid is excessive, an apparatus becomes dirty not only during replacing the sample substrate, but also when the sample substrate moves as the prism is pressed against the sample substrate. Accordingly, the matching liquid may be attached to an excitation light incident surface of the prism, thereby preventing an evanescent field from being produced. 
         [0009]    In addition, in the method disclosed in JP-A-8-136554, if the amount of flown matching liquid is excessive, when the prism is pressed against the sample substrate or is moved with respect to the sample substrate, the matching liquid flows into the oil puddle and thus the matching liquid between the prism and the sample substrate becomes insufficient, thereby causing air to be easily introduced therebetween. On the other hand, if the amount of flown matching liquid is too small, likewise, air is likely to be introduced between the prism and the sample substrate. Accordingly, since it is required to pay attention to the amount of filling of the matching liquid and the movement of the sample substrate, the disclosed method has a problem of operability as a whole. 
         [0010]    In this manner, the above-described conventional techniques have insufficient consideration of a structure of an apparatus with good operability which is capable of producing a stable evanescent field. 
       BRIEF SUMMARY OF THE INVENTION 
       [0011]    The present invention relates to an analysis technique for qualitatively detecting or quantifying biomolecules by producing an evanescent field on a surface of a substrate, exciting fluorescently labelled biomolecules on the substrate surface in the evanescent field, and detecting the resultant fluorescence emitted from the biomolecules, and a well is provided in a surface opposing a sample substrate, and the matching liquid is filled in the well so as to involve the sample substrate in the matching liquid and fill the matching liquid between the sample substrate and the prism, thereby overcoming the above problem. 
         [0012]    By providing the well in the surface opposing the sample substrate of the prism, it is possible to prevent the matching liquid from being leaked out and preventing an excitation light incidence surface of the prism or an apparatus from being dirty by the leaked matching liquid. In addition, since the well can be filled with the matching liquid by simply pouring the matching liquid into the well, operability can be improved. 
         [0013]    Other objects, features and advantages of the invention will become apparent from the following description of the embodiments of the invention taken in conjunction with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF SEVERAL VIEWS OF DRAWING 
         [0014]      FIG. 1A  is a view illustrating a configuration of Embodiment 1 according to the invention. 
           [0015]      FIG. 1B  is an enlarged perspective view illustrating a prism  101  and its vicinity in Embodiment 1 of the invention. 
           [0016]      FIGS. 2A to 2E  are views illustrating modifications of the prism in Embodiment 1 of the invention. 
           [0017]      FIG. 3  is a view illustrating an angle of evanescent irradiation in Embodiment 1 of the invention. 
           [0018]    FIGS.  4 A 1  to  4 C 1  are views illustrating modifications of a prism supporting member B in Embodiment 1 of the invention. 
           [0019]    FIGS.  4 A 2  to  4 C 2  are projective views viewed in arrow directions in FIGS.  4 A 1  to  4 C 1 . 
           [0020]      FIGS. 5A to 5D  are conceptual views of a real time sequencing method in Embodiment 1 of the invention. 
           [0021]      FIG. 6  is a flow chart of a measuring process in Embodiment 1 of the invention. 
           [0022]      FIG. 7  is a sectional view including an excitation light path in the vicinity of a prism in Embodiment 2 according to the invention. 
           [0023]      FIGS. 8A and 8B  are sectional views including an excitation light path in the vicinity of a prism in Embodiment 3 according to the invention. 
           [0024]      FIG. 9A  is a view illustrating a form of transmission/reflection at an interface between a prism and a matching liquid in Embodiment 3 of the invention. 
           [0025]      FIG. 9B  is a graph illustrating a relation between an incident angle and reflectivity in Embodiment 3. 
           [0026]      FIG. 10A  is an enlarged view of a prism in Embodiment 4 according to the invention. 
           [0027]      FIG. 10B  is a partial sectional view of Embodiment 4 of  FIG. 10A . 
           [0028]      FIG. 10C  is a view illustrating a modification of Embodiment 4. 
           [0029]      FIGS. 11A and 11B  are enlarged views of a prism in Embodiment 5 according to the invention. 
           [0030]      FIG. 12  is an enlarged view of the vicinity of a prism in Embodiment 6 according to the invention. 
           [0031]      FIGS. 13A to 13C  are enlarged views of the vicinity of a prism in Embodiment 7 according to the invention. 
           [0032]      FIGS. 14A and 14B  are enlarged views of the vicinity of a prism in Embodiment 8 according to the invention. 
           [0033]      FIGS. 15A to 15C  are views illustrating modifications of a shape of well formed on a sample substrate in Embodiment 8 of the invention. 
           [0034]      FIG. 16  is a sectional view including excitation light paths from two light sources in the vicinity of a prism when the excitation light share the same path in Embodiment 3 of the invention. 
           [0035]      FIG. 17A  is a view showing light paths after incidence of light into a matching liquid when the excitation light from two light sources share the same path in Embodiment 3 of the invention. 
           [0036]      FIG. 17B  is a graph showing a relation between thickness of a matching liquid and deviation of an evanescent irradiation position when the excitation light from two light sources share the same path in Embodiment 3. 
           [0037]      FIG. 17C  is a table showing parameter values used for calculation in  FIG. 17B . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0038]    Hereinafter, exemplary embodiments of the present invention will be described with reference to the accompanying drawings. 
       Embodiment 1 
       [0039]      FIG. 1A  is a view illustrating a configuration of Embodiment 1 according to the invention.  FIG. 1B  is an enlarged perspective view showing a prism  101  and its vicinity. 
         [0040]    A prism  101  provided with a well is, for example, made of S-BAL14 and has three 60° equilateral surfaces each having a size of 60 mm×50 mm. An acryl frame having height of about 8 mm (depth of the well later), as a wall of the well, is adhered to one (surface opposed to a sample substrate  106 ) of the three surfaces. Aside from the above-described method, a well structure may be prepared by cutting a surface of the prism  101 . 
         [0041]    Height of the well is required to be adjusted such that a surface of the sample substrate  106  including a sample supporting member  105  which opposes the prism  101 , can be immersed in a matching liquid  104 . Instead of S-BAL14, the material of the prism may be glass such Bak4, quartz or the like, resin such as PDMS or the like, or other materials as long as they have low absorptiveness and self-fluorescence to an excitation wavelength. 
         [0042]    There is no problem for the prism to have the shape shown in  FIG. 1  or the shapes  201  to  205  shown in  FIGS. 2A to 2E , but it is required to allow light to be incident from the prism  101  and  201  to  205  into the matching liquid  104  in such a manner that the total reflection condition expressed by the following Equation 1 is satisfied. 
         [0000]      η p &gt;sin −1 ( n   aq   /n   p )   [Equation 1] 
         [0043]    Here, the total reflection condition of Equation 1 will be described with reference to  FIG. 3 . As shown in  FIG. 3 , θ p  represents an incidence angle from a prism  301  into a matching liquid  302 , n aq  represents a refractive index of a sample solution  304 , and n p  represents a refractive index of the prism  301 . In this embodiment, since n p =1.57 for the prism made of the material S-BAL14 and n aq =1.33 for the sample solution  304 , an incidence angle condition for total reflection according to Equation 1 becomes θ p &gt;57.9 degree, and the incidence angle condition is adjusted to meet this condition. If the incidence angle does not meet this condition, excitation light permeates the sample solution  304  without being totally reflected. This leads to deterioration of measurement sensitivity due to background intensity caused by Raman scattering of water, which may result in remarkable deterioration of measurement precision of single fluorescent molecule. Incidentally, the numeral  303  denotes a sample substrate. 
         [0044]    The prism  101  is fixed to a prism driver  103  by a prism supporting member  102  and may be moved in X, Y and Z axis directions by controlling the prism driver  103  manually or automatically by a controller. 
         [0045]    The well of the prism  101  is filled with glycerol as the matching liquid  104 . As the Z axis of the prism driver  103  is moved such that the matching liquid  104  slowly approaches to the sample substrate  106 , the matching liquid  104  is filled between the sample substrate  106  and the prism  101 . Instead of glycerol, the matching liquid  104  may be immersion oil or the like, but preferably has a refractive index close to those of the sample substrate  106  and the prism  101  and low absorptiveness and self-fluorescence to an excitation wavelength. If there is a large difference in refractive index, a loss by excitation light reflection at an interface between the matching liquid  104  and the sample substrate  106  or prism  101  may increase, lowering the excitation strength, or reflected light may become background light in a measurement region by repeated reflection on neighboring members, thereby disturbing a measurement. In addition, if the absorptiveness and self-fluorescence to the excitation wavelength are high, an excitation power is lowered or background intensity increases by the fluorescence, thereby disturbing the measurement. 
         [0046]    The sample substrate  106  is prepared by attaching two substrates together, one being quartz glass of 45 mm×25 mm formed on a side (lower surface) opposing to the prism  101  and another being a PDMS substrate (having the same size as the lower surface) formed with a flow channel on an upper side. The intended solution flows into an observation region within the sample substrate  106  through an inflow path  107  and an outflow path  108  which are combined with both ends of the PDMS substrate for exchange of solution. In addition, aside from the above materials, the sample substrate  106  may employ materials having absorptiveness and self-fluorescence to an excitation wavelength, which should be as low as to have no effect on a measurement. The size of the sample substrate  106  is required to be as small as to be contained in the well or the prism  101  is required to be as large as to contain the sample substrate  106 . 
         [0047]    The sample supporting member  105  is used to tightly press and fix the sample substrate  106  against a sample stage  109 . This can prevent irregular deviation of a fluorescent image due to drift of the sample substrate  105  during measurement. 
         [0048]    In this embodiment, two polycarbonate plates having thickness of 2 mm and size of 35 mm×5 mm are used for the sample supporting member  105 , in each of which drill holes are formed at both ends for passing screws through. The sample substrate  106  is tightly fixed by sandwiching the sample substrate  106  between the sample supporting members and the sample stage and fastening the sample substrate  106  using screwed holes formed in the sample stage  109 . Alternatively, a leaf spring or the like may be used as the sample supporting member  105 . Although this shown embodiment employs a structure where the sample substrate is vertically sandwiched, the sample substrate may be held horizontally by using concave portions or the like formed in the sample supporting members and sandwiching the sample substrate from both horizontal sides in a manner to keep the sample substrate horizontal. 
         [0049]    The sample stage  109  is fixed to the sample driver  110  movable in X, Y and α axis directions. By controlling the sample driver  110  manually, or automatically by a controller  135 , the sample substrate  106  may be scanned in the X and Y axis directions or may be inclined by driving the α axis. The sample driver  110  may be further provided with an additional Z axis for use in focus adjustment in fluorescence observation. Here, the Z axis is an axis perpendicular to a surface of the matching liquid  104  and the X axis is perpendicular to the Z axis and is in parallel to a plane including an excitation light path. The Y axis is perpendicular to an X-Z plane and α is an angle defined between the X axis and the α axis in the X-Z plane. 
         [0050]    Alternatively, the shape of the sample stage  109  may be as denoted by  401  to  403  in FIGS.  4 A 1 - 4 C 2 . However, to avoid a physical interference of an apparatus, when the X and Y axis directions are defined as in  FIG. 1 , an outer width (W (X)SP ) in the X axis direction and an outer width (W (Y)SP ) in the Y axis direction of a supporting member pressing the sample substrate  106  are required to be smaller than the width of the well in the corresponding axial directions, and an inner width (W (X)SPN ) in the X axial direction is required to be larger than an outer diameter of an objective lens. 
         [0051]    Hereinafter, the vicinity of an optical system of  FIG. 1A  will be described. Excitation light emitted from an excitation light source  111  or  112  increases spectrum purity through an excitation filter  113  or  114 , being reflected on a mirror  115  or  116  to become circularly polarized light in a λ/4 plate  141  or  142 , being reflected on an angle adjustment mirror  118  or  117 , being compressed in a condensing lens  119  or  120 , being incident into the prism  101 , and then being incident into the sample substrate  106 . The excitation light incident into the sample substrate  106  is totally reflected at an interface between the sample substrate and a sample aqueous solution to produce an evanescent field on a surface of the sample substrate  106 . Emission from the surface of the sample substrate  106  excited by the evanescent field is collected and collimated in an objective lens  121  and a component (elastic scattering light) having the same wavelength as the excitation light is removed by light emitting filters  122  and  123 . 
         [0052]    Thereafter, the light is transmitted or reflected in different directions for different wavelengths in dichroic mirrors  124  to  126  and focused by imaging lenses  127  to  130  an image is formed on a photoelectric surface of each of image sensors  131  to  134 . Each image obtained in the image sensors  131  to  134  is recorded in a controller  135  serving as a computer having processing, storage and control functions. 
         [0053]    Although an Ar-ion laser having wavelengths of 488 nm and 514.5 nm is used as the excitation light source  111  and a laser diode having a wavelength of about 633 nm is used as the excitation light source  112  in this embodiment, it is to be understood that a second harmonic laser of Nd-YAG, a helium-neon laser or a semiconductor laser may be used as the excitation light source. Although a long pass filter transmitting a wavelength of 525 nm or above is used as the light emitting filter  122  and a notch filter intercepting a wavelength of 620 nm to 645 nm is used as the light emitting filter  123 , it is to be understood that a band pass filter transmitting a range of wavelengths to be detected may be used as the light emitting filter as well. Although, as the dichroic mirrors  124  to  126 , the dichroic mirror  124  is used to transmit wavelengths of 620 nm or above, the dichroic mirror  125  is 560 nm or above, and the dichroic mirror  126  is 690 nm or above, respectively, the dichroic mirrors  124  to  126  having different characteristics may be used to correspond to the excited wavelength and fluorescent dye used. 
         [0054]    In this embodiment, using the configuration shown in  FIGS. 1A and 1B , a DNA sequence is determined in real time by a method as shown in  FIGS. 5A to 5D . An elongation reaction starts by developing a reaction solution containing an enzyme essential for an elongation reaction with fluorescently labelled bases  503  to  506  on a surface of the sample substrate  502  on which a complex  501  of a single DNA strand for determining a sequence and a primer is immobilized ( FIG. 5A ). 
         [0055]    Here, the base  503  is thymine modified with dye emitting infrared fluorescence, the base  504  is adenine modified with dye emitting green fluorescence, the base  505  is cytosine modified with dye emitting red fluorescence, and the base  506  is guanine modified with dye emitting orange fluorescence. By subjecting the substrate to total-internal-reflection illumination with the Ar-ion laser and the laser diode as the excitation light sources  507  and  508 , whenever a base is incorporated in the DNA strand and the complementary strand is elongated, light corresponding to the incorporated base is excited and emitted in the evanescent field on the substrate ( FIG. 5B ). 
         [0056]    When dye is photobleached or separated apart, the fluorescence emission disappears ( FIG. 5C ) and the next base is introduced. By repeating the same process, the extension proceeds ( FIG. 5D ) and, depending on a difference between colors of bright spots emitted at that time, a base sequence is determined by using a difference between strengths of signals input in the image sensors  131  to  134 . 
         [0057]    Hereinafter, a measurement process will be described with reference to a flow chart of  FIG. 6 . 
         [0058]    The prism  101  is separated from the sample stage  109  in advance. The sample substrate  106  is fixed to the sample stage  109  (Step  601 ) and the sample driver  110  drives the sample stage  109  to incline the sample substrate  106  with respect to an opposing surface of the prism  101  with regard to the α axis (Step  602 ). Thus, it becomes difficult for bubbles to be introduced between the matching liquid  104  filled in the well of the prism  101  and the sample substrate  106 . 
         [0059]    Next, the well of the prism  101  is manually filled with the matching liquid  104  (Step  603 ), and the matching liquid  104  in the well slowly approaches to the inclined sample substrate  106  using the Z axis of the prism driver  103  until the matching liquid  104  contacts the sample substrate  106  (Step  604 ). Alternatively, the filling of the matching liquid  104  may be carried out by opening/closing of a valve of a mechanism for injecting/discharging the matching liquid  104  in/from the prism  101 , as will be described later in Embodiment 6. 
         [0060]    In addition, the prism driver may include a coarse motion mechanism used when the matching liquid  104  is distant from the sample substrate  106  and a fine motion mechanism used when the matching liquid  104  is close to the sample substrate  106  for improvement of operability. In this case, position detecting means such as a sensor may be provided to detect a distance from a surface of the prism  101  or a surface of a matching liquid  104  layer to the sample substrate  106 , and the controller  135  may perform control of switch to the fine motion mechanism when it becomes close to a predetermined position. Although it is illustrated here that the matching liquid  104  contacts the sample substrate  106  by the prism driver  103 , these mechanisms may be provided in the sample driver  110 . 
         [0061]    Thereafter, the sample substrate  106  inclined from the α axis of the sample driver  110  is returned to be horizontal with respect to the matching liquid  104  (Step  605 ). Thus, the matching liquid  104  is equally filled on the opposing surface of the sample substrate  106 . A series of driving operations by the sample driver  110  and the prism driver  103  is automatically performed by the controller  135 . 
         [0062]    Next, an operation of immobilizing a complex of a DNA single strand and a primer on the substrate (Step  606 ) using a biotin-avidin binding is performed as follows. A container filled with tris-buffer including biotinylated BSA is equipped as an inflow container  136 , and the surface of the sample substrate  106  is coated with the biotinylated BSA by flowing the solution from an inflow path  107  to an outflow path  108 . A surplus solution coming out of the outflow path  108  is stored in a waste solution container  137 . In order to remove floating biotinylated BSA not coated on the surface of the sample substrate, the inflow container  136  is replaced with a tris-buffer solution container and a cleaning operation to flow a sufficient amount of solution is performed. Hereinafter, likewise, a streptavidin solution and a complex solution of a biotinylated DNA main chain labelled with dye emitting red light and a primer are flown in order and are immobilized. After flowing the solutions, a cleaning operation is performed (Step  606 ). 
         [0063]    For observation of fluorescence of single molecules, shutters  139  and  140  are opened to subject the sample substrate  106  to total-internal-reflection illumination with the Ar-ion laser and the laser diode (Step  607 ), and a fluorescence image of the image sensors  131  to  134  projected in the controller  135  is focused using a driver  138  of the objective lens driver  138  while viewing the fluorescence image (Step  608 ). The sample substrate  106  is moved to a region to be observed using the X and Y axes of the sample driver  110  (Step  609 ) and a position of the complex of the DNA single strand and the primer from an emission bright spot is checked. A reaction starts by introducing a reaction solution containing a fluorescently labelled base and an enzyme from the inflow container  136  (Step  610 ), and at the same time, a base extension reaction is measured in real time by continuously recording 5000 image data in the controller  135  at a frame rate of 10 frames/sec. 
         [0064]    When the sample substrate  106  is taken out, the prism  101  is slowly moved downward using the Z axis of the prism driver  103  until the matching liquid  104  is detached from the sample substrate  106 . At this time, by inclining the sample substrate  106  from the α axis of the sample stage  109 , the matching liquid  104  attached to the surface of the sample substrate  106  can be easily taken out using surface energy of the matching liquid  104  when the prism  101  is moved downward. After detaching the prism  101 , the detached state is maintained for a certain period of time, or a portion of the substrate is somewhat dipped in the liquid surface to absorb liquid drops by a surface tension of the matching liquid, or after confirming that the matching liquid  104  does not flow out of the sample substrate  106  and returning the α axis to the horizontal position, the sample substrate  106  is detached from the sample stage  109  and a new sample substrate  106  is mounted on the sample stage  109 . 
         [0065]    Although it is illustrated here that the matching liquid  104  is detached from the sample substrate  106  using the prism driver  103 , the matching liquid  104  may be detached from the sample substrate  106  using the sample driver  110 . 
         [0066]    Although one flow channel is provided in the sample substrate in this embodiment, a plurality of samples may be continuously measured when the inflow path  107  and the outflow path  108  are provided in each of a plurality of flow channels and the Steps  606  to  612  are repeated. 
         [0067]    With the structure of Embodiment 1, by improving the operability of the matching liquid, difficulty in measurement related to the matching liquid operation can be overcome, the stable measurement can be repeated, and time taken from measurement preparation to measurement completion can be shortened. 
       Embodiment 2 
       [0068]    Next, Embodiment 2 shows an example of measurement by a high aperture-oil immersion type objective lens. 
         [0069]    A fluorescent signal from a single fluorescent molecule is weak. Thus, if a background is high or noise derived from an apparatus is large, it is difficult to detect emission bright spots. Therefore, detecting means having higher sensitivity, such as an objective lens with high numerical aperture, is required. 
         [0070]    However, since an objective lens with numerical aperture of 1.3 or above is required to be used in an oil immersion state, there is a need to fill the immersion oil between a sample substrate and the objective lens. At that time, operability is poor, immersion oil flows around to make an apparatus dirty, or it takes a long time for a filling operation. Further, when the determination in Embodiment 1 is automated, an immersion oil filling device is required, thereby increasing the burden in terms of space and cost. 
         [0071]    Embodiment 2 of the present invention provides means to overcome the above problem. Embodiment 2 has substantially the same basic configuration as Embodiment 1. 
         [0072]      FIG. 7  is a sectional view including an excitation light path in the vicinity of the prism of  FIGS. 1A and 1B . A well is filled with immersion oil used for an oil immersion type objective lens  703  and the immersion oil is also used as a matching liquid  702  for both a sample substrate  704  and a prism  701 . 
         [0073]    As shown in  FIG. 7 , in order to immerse the sample substrate  704  and a lead end of the objective lens  703  in the matching liquid  702 , the sample substrate  704  is pushed and fixed to a sample stage  706  by means of a sample supporting member  705 , as in Embodiment 1, the prism  701  filled with the matching liquid  702  approaches to the sample substrate  704  until the prism  701  contacts the sample substrate  704 , and the matching liquid  702  is supplied from a matching liquid container  708  through a nozzle  709  fixed to the objective lens  703  by opening a small valve  707 . Although the sample substrate  704  may be immersed by continuously moving the prism  701  upward without supply of the matching liquid  702  through the nozzle  709 , further movement of the prism is difficult due to surface energy around the matching liquid  702  and the sample substrate  704 , which may result in poor operability. 
         [0074]    Accordingly, this embodiment provides a mechanism in which the prism  701  is moved upward in the operation described in Embodiment 1, it is checked that the matching liquid  702  contacts the sample substrate  704  without mixture of bubbles, and then the matching liquid  702  is supplied from the nozzle  709  provided above the sample substrate  704 . Although it is shown here that the matching liquid  702  is supplied through the nozzle  709  after the matching liquid  702  contacts the sample substrate  704 , the matching liquid  702  may be supplied before the matching liquid  702  contacts the sample substrate  704 . In this case, however, since the amount of the matching liquid  702  used increases before the contact compared to after the contact, a running cost increases. In addition, if the bubbles should be mixed by the supply of the matching liquid  702  from the nozzle  709 , it means that the upper surface of the sample substrate  704  already gets wet with the matching liquid  702 , and thus it is difficult to check this mixing of the bubbles by visual observation. 
         [0075]    With the apparatus of this embodiment, by doubling as the immersion oil for the objective lens  703  and the matching liquid  702  for the sample substrate  704  and the prism  701 , the operability of the immersion oil becomes improved and a high sensitivity measurement in the high aperture objective lens becomes possible. 
       Embodiment 3 
       [0076]    In Embodiment 1, the excitation light sources  111  and  112  are provided in different light paths and the excitation light is incident from both sides of the prism  101 , as shown in  FIG. 1A . If a dichroic mirror  1602  is placed before a mirror  1601  and it is possible to make excitation light paths from two light sources  1603  and  1604  equal, as shown in  FIG. 16 , it is possible to reduce costs of optical components such as a condensing lens  1609 , mirrors and so on and make an apparatus compact. 
         [0077]    However, if the excitation light is incident from the same plane of a prism  1605  with the above equal light paths, deviation of a position of evanescent irradiation from light sources having different wavelengths increases due to a refraction angle difference at an interface between the prism  1605  and the matching liquid  1606  depending on a wavelength, along with a distance between the sample substrate  1607  and the prism  1605 . 
         [0078]      FIG. 17A  illustrates respective light paths of when excitation light having wavelengths λ 1  and λ 2  transmits a prism  1701  in the same light path, the excitation light is incident into a matching liquid  1702  at an incidence angle θ p  satisfying the conditional Equation 1, and the incident light is totally reflected at an interface between a sample substrate  1703  and a sample solution  1704 . Here, since the excitation light travels in parallel to an X-Z axial plane, a deviation occurs in only the X axis direction. 
         [0079]    In  FIG. 17A , the reason why the two excitation lights travel along different light paths after being incident into the matching liquid  1704  is that there is a difference in refractive index between the prism  1701 , the matching liquid  1702  and the sample substrate  1703  depending on the wavelength, that is, there is a difference in refraction angle therebetween. In  FIG. 17A , assuming that an incidence position and an incidence angle θ p  at the excitation wavelength λ 1  from the prism  1701  to the matching liquid  1702  are the same as those at the excitation wavelength λ 2 , a deviation Δl of an evanescent irradiation position is obtained by the sum of a deviation Δl m  occurring when the excitation light transmits the matching liquid  1702  and a deviation Δl s  occurring when the excitation light transmits the sample substrate  1703  according to the following Equation 7: 
         [0000]      Δ l=Δl   m   +Δl   s    [Equation 7] 
         [0080]    Where, in order to obtain Δl m  and Δl s , refractive indexes at the prism  1701 , the matching liquid  1702  and the sample substrate  1703  of the excitation light having the wavelengths λ 1  and λ 2  are defined as n p (λ 1 ), n p (λ 2 ), n m (λ 1 ), n m (λ 2 ), n s (λ 1 ), and n s (λ 2 ), respectively, and thicknesses of the matching liquid  1702  and the sample substrate  1703  are defined as Δ m  and Δ s , respectively (see  FIG. 17A ). At this time, transmission optic angles θ m1  and θ m2  of the excitation light having the wavelengths λ 1  and λ 2  from the prism  1701  to the matching liquid  1702  are given according to Snell&#39;s law, as follows: 
         [0000]      θ m1 =sin −1 (( n   p (λ 1 )/ n   m (λ 1 ))sin(θ p ))   [Equation 8] 
         [0000]      θ m2 =sin −1 (( n   p (λ 2 )/ n   m (λ 2 ))sin(θ p ))   [Equation 9] 
         [0081]    Accordingly, Δl m  is expressed as follows: 
         [0000]      Δ l   m   =Δm ×|tan(θ m1 )−tan(θ m2 )|  [Equation 10] 
         [0082]    Likewise, arranging Equations 8 to 10, Δl s  is expressed as follows: 
         [0000]      Δ l   s   =Δs ×|tan(sin −1 (( n   m (λ 1 )/ n   s (λ 1 ))sin(θ m1 )))−tan(sin −1 (( n   m (λ 2 )/ n   s (λ 2 ))sin(θ m2 )))|  [Equation 11] 
         [0083]    Accordingly, putting values of Equations 10 and 11 into Equation 7, the deviation Δl of the evanescent irradiation position can be obtained. 
         [0084]      FIG. 17B  shows a plotting diagram of the deviation Δl of the evanescent irradiation position calculated with respect to thickness Δm of the matching liquid  1702  when excitation light having wavelengths λ 1 =488 nm and λ 2 =633 nm is incident into the general prism  1701  made of S-BAL14, quartz or BK7. Parameter values used for the calculation are listed in a table of  FIG. 17C . In the table, the incidence angle θ p  is the maximum value (critical angle) satisfying the Equation 1. 
         [0085]    A case where the two excitation light paths in Embodiment 1 are arranged to be the same as shown in  FIG. 16  is considered. Since the thickness of the sample supporting member  1608  (thickness of a screw head portion and a polycarbonate plate) is about 3.5 mm, a distance between the sample substrate  106  and the prism  1605  is a minimum of 4 mm. In this case, according to  FIG. 17C , the deviation of evanescent irradiation positions of two excitation lights for any material of the prism  1701  becomes about 0.3 mm or above. As a condition for confirming two evanescent irradiation positions in a measurement field of view, the above position deviation is required to be smaller than the sum of an irradiation region diameter and a field of view size. Since a field of view size is about φ100 μm and an irradiation region diameter is 120 μm in Embodiment 1, the condition becomes 0.22 μm or below. Accordingly, if there occurs an irradiation position deviation of about 0.3 mm or above, the minimum of one evanescent irradiation position is out of the field of view. Accordingly, although an evanescent irradiation region is required to be moved by adjusting an angle of the light sources  1603  and  1604  or the mirror  1615 , since the irradiation region can not be viewed by visual observation if the evanescent irradiation position is out of the field of view, it is very difficult to adjust an optical axis. In other words, if irradiation is made from the same path as shown in  FIG. 16  in the shape of the prism in Embodiment 1, it may take much time for adjustment of the optical axis. 
         [0086]    Another problem is a reflected component of the excitation light, which is produced at an interface between the prism  101 , the matching liquid  104  and the sample substrate  106 . As a difference in refractive index between these three materials increases, a loss by reflection increases, which may result in reduction of excitation strength. In addition, if strength of reflected light is high, reflection repeated around the prism may enter an observation region, which may result in increase of background light and hence reduction of measurement sensitivity. Nonetheless, although refractive indexes of the prism  101 , the matching liquid  104  and the sample substrate  106  are required to be similar to one another, the material of the sample substrate  106  is limited to those such as synthetic quartz which does not emit fluorescent light for an excited wavelength. 
         [0087]    Accordingly, not only is the material of the prism  101  limited, but also a manufacture cost may increase such as in the case of synthetic quartz. In the present invention, since a material with a size as large as to contain the sample substrate  106  is used, the manufacture cost problem cannot be ignored. 
         [0088]    This embodiment provides a prism configuration to overcome the above two problems. This embodiment has substantially the same basic configuration as Embodiment 1.  FIG. 8A  is a sectional view including excitation light paths around the prism of  FIG. 1 . An inclined plane in parallel to an outer side of a prism  801  is provided in the bottom of a well. An angle of the inclined plane is set such that excitation light is totally reflected on a surface of a sample substrate  802  when the excitation light is incident perpendicular to the inclined plane. That is, the angle β of the inclined plane, defined in  FIG. 8B , satisfies the following Equation 2. 
         [0000]      β&gt;sin −1 ( n   aq   /n   m )   [Equation 2] 
         [0089]    Where, n aq  is a refractive index of a sample solution  803  and n m  is a refractive index of a matching liquid  804 . With the configuration of this embodiment, since the excitation light is incident perpendicular to the inclined plane of the prism  801  and the well bottom, there occurs no deviation of light paths depending on a wavelength at the interface even if a plurality of kinds of light sources are mixed in the same light path. In this embodiment, as a mechanism for confirming the perpendicular incidence of the excitation light, an aperture iris  809  having a diameter aperture substantially as the same as a diameter of the excitation light is disposed at a light source side of a condensing lens  808 . If the excitation light is perpendicularly incident, since the reflected light incident into the surface of the prism  801  returns to the direction of the light source substantially in the same light path as the light incidence, the returning light beamed on the aperture iris  809  can be confirmed. According to the above method, since the evanescent irradiation region can be put near the field of view of the objective lens  807 , an evanescent region can be set within the field of view only by finely adjusting an angle of the light sources  1603  and  1604  or the mirror  1615 . Although it is shown here that the aperture iris  809  is disposed at the light source side of the condensing lens  808 , the aperture iris  809  may be disposed at the light source side of the prism or mirror  1601 . In addition, since the perpendicularity of the light incidence is an indicator to alleviate the trouble of positioning of the evanescent irradiation region, if the incidence angle is deviated from the perpendicularity, the inherent effect of this embodiment does not disappear. 
         [0090]    Next, an effect of the reflected light will be described. 
         [0091]      FIG. 9A  shows a light incidence angle θ i  and a light transmission angle θ t  from a prism  901  having a refractive index n p  to a matching liquid  902  having a refractive index n m . 
         [0092]    According to Fresnel&#39;s formula, reflectivities of p-polarization whose electric field vector is in parallel to an incidence plane and s-polarization whose electric field vector is perpendicular to the incidence plane are as follows:
   i) If θ i ≠0,   
 
         [0000]      ( p -polarization reflectivity)=(sin(θ i −θ t )/sin(θ i +θ t )) 2    [Equation 3] 
         [0000]      ( s -polarization reflectivity)=(tan(θ i −θ t )/tan(θ i +θ t )) 2    [Equation 4]   ii) If θ i =0,     
         [0000]      ( p/s -polarization reflectivity)=(( n   1   −n   2 )/( n   1   +n   2 )) 2    [Equation 5] 
         [0000]    Where, the light transmission angle θ t  is obtained as follows according to Snell&#39;s law. 
         [0000]        n   p  sin θ i   =n   m  sin θ t    [Equation 6] 
         [0095]    Therefore, according to Equations 3 to 6, the reflectivity when the matching liquid  902  is glycerol (refractive index n m =1.47) and the material of the prism  901  is S-BSL14 (refractive index n p =1.57) is as shown in  FIG. 9B , as in Embodiment 1. As a result, when the light is perpendicularly incident from the prism  901  into the matching liquid  902  (θ i =0), reflected light becomes minimal and then slowly increases. 
         [0096]    When the reflected light enters an observation region, its strength is required to be limited to 1 μW or below. Since a minimum of two times of reflection on the surface of the prism is required, when reflectivity to allow light strength to be 0.3 μW or below at these two times of reflection is assumed as an allowable range, since incidence light power of the general single molecule fluorescence measurement is only 50 μW, it is believed that reflectivity of 0.2% or below is acceptable. 
         [0097]    An allowable range of the incidence angle θ i  when the material of the prism  901  of  FIG. 9B  is S-BLS14 and the matching liquid  902  is glycerol is 0 to 31 degrees. An allowable range of θ i  when the material of the prism  901  is BK7 (n p =1.52) is 0 to 53 degrees. An allowable range of θ i  when the material of the prism  901  is quartz (n p =1.46) and the matching liquid  902  is immersion oil (n m =1.52) is 0 to 45 degrees. Since any of these specified angles is an incidence angle from the prism  901  into the matching liquid  902 , when this incidence angle is changed to an incidence angle from air into the prism  901 , an allowable range satisfying the above three combinations becomes 0 to 54 degrees. It is here noted that the total reflection condition of Equation 1 is required to be considered. 
         [0098]    Next, an allowable angle of parallism of the well bottom of the prism and the excitation light incidence plane will be described by way of an example of a prism made of S-BAL14 which has three 60° equilateral surfaces each having a size of 60 mm×50 mm, which was used in Embodiment 1. When the excitation light is perpendicularly incident, all of three β angles in  FIG. 8B  are 60 degrees. Here, for the sake of convenience, for the three β angles, it is assumed that an angle formed between the excitation light incidence plane and the matching liquid surface is β 1 , an angle formed between the well bottom of the prism and the matching liquid surface is β 2 , and an incidence angle of the excitation light into the sample substrate is β 3 . An allowable angle of β 1  when glycerol (refractive index: 1.47) is used as the matching liquid and the excitation light is perpendicularly incident into the prism is obtained. Here, it is assumed that β 2 =60 degrees (constant). First, when β 1  decreases by Δβ 1 (−) degree, an allowable angle is considered. Since β 3 &gt;57.9 degrees according to the total reflection condition of Equation 1, it is established that the incidence angle θ i  from the prism into the matching liquid is smaller than 1.97 degrees according to Snell&#39;s law of Equation 8 or 9. Next, an allowable angle of the perpendicular incidence is considered. According to the allowable area of the above-obtained incidence angle θ i  to neglect an effect of the reflected light when S-BSL14 is used for the prism  901  and glycerol is used as the matching liquid  902 , it is obtained that Δβ 1 (+)&lt;31 degrees. From the above, an allowable angle of deviation of the parallism of the well bottom of the prism and the excitation light incidence plane becomes −1.97 to +31 degrees. 
         [0099]    As described above, with the configuration of this embodiment, although the best effect is obtained when the well bottom of the prism is in substantial parallel to the excitation light incidence plane to satisfy the above parallism condition and the excitation light is perpendicularly incident, the excitation light may be incident in a range of 0 (perpendicular) to 54 degrees with respect to the light incidence plane of the prism even if the light is deviated from the perpendicularity. It is here noted that the total reflection condition of the Equation 1 is required to be considered. In addition, deviation of the parallism of the well bottom of the prism and the excitation light incidence plane is preferably −2 to +30 degrees. Under such a condition, since the prism material is not required to meet a refractive index of the matching liquid and thus a width of selection of the material is widened, the manufacture cost can be reduced. 
         [0100]    Accordingly, it can be seen that the two above-mentioned problems can be overcome by this embodiment in which the inclined plane in substantial parallel to the outer side of the prism  801  is provided in the well bottom and the excitation light is incident substantially perpendicular to the inclined plane. This embodiment may be incorporated into Embodiment 1 or 2. 
       Embodiment 4 
       [0101]    Embodiment 4 of the present invention has substantially the same basic configuration as Embodiment 1 except that projections  1002  are provided in a well bottom of a prism  1001  as shown in  FIG. 10A . The projections  1002  can be used as a guide when the prism  1001  approaches to a sample substrate to adjust a relative position therebetween. In addition, since the prism  1001  and the opposing surface of the sample substrate can be maintained in parallel with good reproducibility, it is possible to suppress variation of evanescent irradiation strength due to deviation of a relative angle therebetween. In addition, it is possible to prevent close adhesion of the sample substrate and the prism and thus facilitate detachment of the prism from the sample substrate. 
         [0102]    Although the projections are prepared by adhering pillar-shaped PMDSs, as denoted by  1002  in  FIG. 10B , to the surface of the prism, with their height adjusted as the same, the projections may be two hog-backed projections, as denoted by  1003  in  FIG. 10C , or doughnut-shaped projections lacking a part. In a case of doughnut-shaped projections, if their sections are completely circular, since air may be introduced between the matching liquid and the sample substrate when the matching liquid is filled, thereby making a measurement difficult, the doughnut-shaped projections preferably have a section similar to that of the projections of  FIG. 10B . In addition, adhesion portions  1004  of the projections  1002  are curved to make bubbles difficult to be attached, as shown in an enlarged view of  FIG. 10A . The material for the projections may be hard plastic such as acryl as well as elastic resin such as PDMS. This embodiment may be incorporated into Embodiment 1 or 2. 
       Embodiment 5 
       [0103]    Embodiment 5 of the present invention has substantially the same basic configuration as Embodiment 1 except that a liquid leakage prevention groove  1102  is provided in a wall forming a well of a prism  1101  as shown in  FIG. 11A . Even if the prism  1101  is inclined or impacted with the prism  1101  filled with a matching liquid  1103  and thus the matching liquid  1103  flows out along the surface of the prism  1101 , the matching liquid  1103  can be stopped in the groove  1102 . As a structure to achieve the same effect, a liquid leakage preventing step  1105  such as prism  1104  as shown in  FIG. 11B  may be provided. In the preparation, although two small and large rectangular PDMS frames are mounted on a plane of the prism  1101 , plastic material such as acryl or glass material may be processed into a shape of frame and may be used. Of course, the plane of the prism  1101  may be cut to replace the frames. This embodiment may be incorporated into any of Embodiments 1 to 4. 
       Embodiment 6 
       [0104]    Embodiment 6 of the present invention has substantially the same basic configuration as Embodiment 1 except that an inlet  1203  and an outlet  1204  for exchanging matching liquid  1202  are provided in a wall forming a well of a prism  1201 , as shown in  FIG. 12 . Since the inlet  1203  is connected to a matching liquid container  1205  storing a new matching liquid, the matching liquid may flow to exchange the matching liquid  1202  in the well by opening/closing a valve  1206 . The used matching liquid  1202  is stored in a waste matching liquid container  1207 . The inherent effect of this embodiment is to simplify exchange of an old matching liquid. This embodiment may be incorporated into any of Embodiments 1 to 5. 
         [0105]    When this embodiment is incorporated into Embodiment 2, the same matching liquid container as the matching liquid container  708  and  1205  may be used. 
       Embodiment 7 
       [0106]    Embodiment 7 of the present invention has substantially the same basic configuration as Embodiment 1.  FIG. 13A  shows a prism  1301  and its vicinity. This embodiment includes a temperature adjusting unit  1303  for adjusting temperature of a matching liquid  1302 . The temperature of the temperature adjusting unit  1303  is adjusted by a temperature controller  1304  and the temperature of the matching liquid  1302  may be monitored by a temperature sensor  1305 . The temperature controller  1304  may be also used as the controller  135  in Embodiment 1. In the temperature adjusting unit  1303 , a heat generating film formed of an ITO film having thickness of about 0.3 mm, which can be heated up to 70° C., is attached to a well bottom of the prism  1301 . In addition, the temperature adjusting unit  1303  may be in the form of a wire such as a nichrome wire or strip. In addition, the temperature of the matching liquid  1302  may be adjusted by sandwiching the prism  1301  at both sides with a temperature adjusting unit  1306 , as shown in  FIG. 13B , or by circulating the matching liquid  1302  within a temperature adjusting unit  1307  by connecting a pipe to wall surfaces of both ends of the well, as shown in  FIG. 13C . The inherent effect of this embodiment is to control temperature with little unevenness by controlling the temperature of a sample substrate through the matching liquid. Particularly, in a case of elongation reaction as in Embodiment 1, although reaction temperature is required to increase up to 70° C. in order to raise activity of an enzyme, an elongation reaction efficiency is varied depending on a position of the sample substrate if there is unevenness in temperature, which may result in a serious problem due to remarkable deterioration of a measurement efficiency. For that account, this embodiment aims at and can achieve high throughput of a real time DNA sequencing method. This embodiment may be incorporated into any of Embodiments 1 to 6. 
       Embodiment 8 
       [0107]    Embodiment 8 of the present invention has substantially the same basic configuration as Embodiment 1 except that a well retaining a matching liquid  1402  is formed on a sample substrate  1401  fixed to a sample stage  1408  in a sample supporting member  1410  by adding a matching liquid holding member  1403 . In addition, in this embodiment, no well is provided on a prism  1404  side.  FIG. 14B  is a sectional view including an excitation light path of  FIG. 14A . The prism  1404  is immersed in the matching liquid  1402  retained in the well and then the same measurement as Embodiment 1 is performed. In this embodiment, since a well surface opposing to the prism  1404  is required to be a top surface, an objective lens  1405  opposing to a fluorescence detection side is arranged below the sample substrate. Other components have the same relative position relation as those as described above except that positions of components of an irradiation system and a detection system are vertically reversed. The sample stage  1408  is driven by a sample driver  1409 . 
         [0108]    The sample substrate  1401  is prepared by together attaching two substrates, one being quartz glass of 50 mm×40 mm formed on a surface (upper surface) opposing to the prism  1404  and another being a PDMS substrate (having the same size as that of the upper surface) formed with a flow channel on a lower surface. In addition, instead of the above materials, the sample substrate  1401  may employ materials having absorptiveness and self-fluorescence to an excitation wavelength, which are as low as to have no effect on a measurement. The well retaining the matching liquid  1402  is prepared by adhering matching liquid holding members  1403  and  1502 , which are formed of an acryl frame having a surface of 25 mm×25 mm and a height of 5 mm and serve as a wall of the well, on the sample substrate  1501 , as shown in  FIG. 15A . The material of the matching liquid holding members  1403  and  1502  may be hard plastic as well as soft resin such as silicon or PDMS. In addition, the well may be formed by adhering the matching liquid holding members  1403  and  1502  having a vessel shape as shown in  FIG. 15B  to the sample substrate  1401  and  1501  or by providing a puddle by cutting the sample substrates  1401  and  1501  as shown in  FIG. 15C . Although this embodiment uses a 60° equilateral prism made of S-BAL14 and having a surface of 10 mm×15 mm as the prism  1404 , a size as large as to be contained in the well and the material may be any glass such Bak4, quartz or the like, resin such as PDMS or the like, or other materials as long as they have low absorptiveness and self-fluorescence to an excitation wavelength. The inherent effect of this embodiment is to reduce a prism manufacture cost since this embodiment can provide a prism smaller than that of Embodiment 1. 
         [0109]    The present invention can be applied to a DNA sequencer using an extension reaction, a DNA micro array reader of a total reflection fluorescent type, etc. 
         [0110]    It should be further understood by those skilled in the art that although the foregoing description has been made on embodiments of the invention, the invention is not limited thereto and various changes and modifications may be made without departing from the spirit of the invention and the scope of the appended claims.