Abstract:
An apparatus for determining directional transport processes with a scanning microscope ( 100 ) is disclosed. A deflection means ( 5 ) for coupling in an illuminating light beam ( 3 ), and a scanning module ( 7 ) for graphical display of a specimen ( 15 ) on a peripheral device ( 27 ) and for positioning the illuminating light beam ( 3 ) for a specific time period on a location of the specimen ( 15 ), are provided. Provided between the deflection means ( 5 ) and the scanning module ( 7 ) is a device ( 65 ) that generates, from the illuminating light beam ( 3 ), at least two illuminating light beams ( 3   a ,    3   b ) that merge at a rotation point ( 70 ) of the scanning module ( 7 ). Each of the several illuminating light beams ( 3   a   , 3   b ) generates a respective focus ( 72 ) in the specimen ( 15 ), all the foci ( 72 ) being arranged in one plane ( 75 ).

Description:
RELATED APPLICATIONS 
   European Patent EP 0 941 470 discloses a system in which a Fluorescence Correlation Spectroscopy (FCS) module is associated with an imaging scanning microscope. The FCS module is coupled directly onto the scanning microscope. The light for FCS examination is coupled out of the detection beam path of the scanning microscope and conveyed to the FCS module. Unequivocal detection of the diffusion direction is not possible with this system. 
   FIELD OF THE INVENTION 
   The invention concerns an apparatus for determining directional transport processes. 
   The invention furthermore concerns a method for determining directional transport processes. The invention concerns in particular a method for determining directional transport processes using a scanning microscope that encompasses a deflection means for coupling in an illuminating light beam and a scanning module for graphical display of a specimen on a peripheral device and for positioning the illuminating light beam for a specific time period on a location of the specimen. 
   BACKGROUND OF THE INVENTION 
   Fluorescence correlation spectroscopy, in its implementation in a microscope assemblage, is suitable for the investigation not only of non-directional molecular diffusion, but also of directional transport processes, such as particle flow, that may be overlaid on the diffusion. The concentration and diffusion coefficient of correspondingly labeled molecules, and the absolute value of their flow velocity can be derived from autocorrelation analysis of the fluorescence signal of the molecules. The radial symmetry of the focus about the optical axis makes it impossible, however, to determine the direction of the flow. (Publications: Brinkmeier (2001) in: Fluorescence Correlation Spectroscopy—Theory and Application, pp. 379–395, eds.: R. Rigler, E. Elson, Springer-Verlag, Heidelberg/Berlin; Dittrich &amp; Schwille (2002): Anal. Chem. 74, 4472). 
   European Patent EP 0 941 470 discloses a system in which an FCS module is associated with an imaging scanning microscope. The FCS module is coupled directly onto the scanning microscope. The light for FCS examination is coupled out of the detection beam path of the scanning microscope and conveyed to the FCS module. Unequivocal detection of the diffusion direction is not possible with this system. 
   SUMMARY OF THE INVENTION 
   It is the object of the invention to create an apparatus with which diffusion processes in a specimen or a sample can be determined and evaluated. 
   The object is achieved by way of an apparatus comprising: 
   a deflection means for coupling in an illuminating light beam, 
   a scanning module for scanning the illuminating light beam across a specimen, for graphical display of the specimen on a peripheral device and for positioning the illuminating light beam for a specific time period on a location of the specimen, 
   a first device for generating at least two illuminating light beams, wherein the device is provided between the deflection means and the scanning module, and 
   a rotation point is defined on the scanning module in which the illuminating light beams merge and wherein each of the illuminating light beams defining a focus in the specimen that are all arranged in one plane. 
   It is the object of the invention to create a method with which diffusion processes in a specimen or a sample can be determined and evaluated. 
   The aforesaid object is achieved by way of a method comprising the steps of: 
   generating at least two illuminating light beams from the illuminating light beam, by a device being provided for that purpose between the deflection means and the scanning module; 
   combining the illuminating light beams at a rotation point of the scanning module; and 
   defining a respective focus for each illuminating light beam, all the foci being arranged in one plane. 
   It is particularly advantageous for detection of the diffusion direction if two foci, slightly shifted with respect to one another, are used (bifocal configuration). The absolute value of the flow velocity can be determined from a cross-correlation of the signals from these foci. The flow direction can be ascertained by rotating the connecting line between the foci about the optical axis or the direction defined by the illuminating light beam. The disadvantage of the existing art is the complexity of configuring and aligning two confocal beam paths shifted slightly with respect to one another. Adjustment of the spacing, as well as rotation, are also laborious. Integration into, for example, existing confocal laser scanning microscopes is difficult if not impossible. 
   It is particularly advantageous if the scanning microscope is equipped with a deflection means for coupling in an illuminating light beam. It has proven to be particularly convenient and user-friendly if the deflection means is configured in the form of an AOBS™. Also provided is a scanning module with which a graphical display of a specimen on a peripheral device is also possible. The scanning module can also be used to position the illuminating light beam for a specific time period on a location of the specimen. The at least one location can be selected by the user, for example, on the display using a mouse. Provided between the deflection means and the scanning module is a device that generates, from the illuminating light beam, at least two illuminating light beams that merge at a rotation point of the scanning module. Respective corresponding foci that are all arranged in one plane are then defined in the specimen by way of the several illuminating light beams. 
   The device encompasses a neutral or dichroic beam splitter that splits the illuminating light beam into a first and a second illuminating light beam. A displaceable and/or pivotable deflection mirror is associated with one of the illuminating light beams. With the displaceable and/or pivotable deflection mirror, it is possible to perform an adjustment such that the illuminating light beams split by the beam splitter merge at the rotation point of the scanning module. To eliminate undesirable scattered radiation, a beam trap is associated with the beam splitter opposite the deflection mirror. 
   The device is furthermore equipped in such a way that the detected light beams proceeding from the specimen are combined into a single detected light beam. The detected light beam is directed, by a beam splitter provided in a housing, onto a first and a second detection channel. Downstream from the first and the second detection channel is a respective multimode fiber that conveys the detected light beam to a respective avalanche photodiode. The avalanche photodiodes are accommodated together in a housing in order to ensure sufficient cooling and light-tightness for the avalanche photodiodes. 
   The device provided in the apparatus according to the present invention is furthermore arranged in a housing. The device is rotatable about a rotation axis, the rotation axis coinciding with the direction defined by the illuminating light beam and the detected light beam. 
   Further advantageous embodiments of the invention are evident from the dependent claims. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The subject matter of the invention is depicted schematically in the drawings and will be described below with reference to the Figures, in which: 
       FIG. 1  schematically depicts a scanning microscope having an SP module; 
       FIG. 2  schematically depicts the arrangement of the device components for FCS analysis in a scanning microscope; 
       FIG. 3   a  schematically depicts the device for bifocal illumination of a specimen for FCS analysis; 
       FIG. 3   b  shows the arrangement of the focal volumes in the specimen in a plan view; 
       FIG. 3   c  shows the arrangement of the focal volumes in the specimen, depicting a cross section through the sample or specimen; 
       FIG. 4   a  schematically depicts the components for bifocal illumination of a specimen for FCS analysis with more than two foci; 
       FIG. 4   b  shows the arrangement of the focal volumes in the specimen in a plan view; 
       FIG. 4   c  shows the arrangement of the focal volumes in the specimen, depicting a cross section through the sample; and 
       FIG. 5  shows the correlation functions for the extreme cases of 0° (diamonds) and 90° (squares) and the difference (triangles), for an autocorrelated diffusion correlation time of 1 msec. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     FIG. 1  shows the schematic configuration of a confocal scanning microscope  100  in which the apparatus according to the present invention is used. Illuminating light beam  3  coming from at least one illumination system  1  is directed by a beam splitter or a suitable deflection means  5  to a scanning module  7 . Before illuminating light beam  3  strikes deflection means  5 , it passes through an illumination pinhole  6 . Scanning module  7  encompasses a gimbal-mounted scanning mirror  9  that guides illuminating light beam  3 , through a scanning optical system  12  and a microscope optical system  13 , over or through a specimen  15 . In the case of non-transparent specimens  15 , illuminating light beam  3  is guided over the specimen surface. With biological specimens  15  (preparations) or transparent specimens, illuminating light beam  3  can also be guided through specimen  15 . For these purposes, non-luminous preparations are prepared, if applicable, with a suitable dye (not depicted, since it is established existing art). The dyes present in the specimen are excited by illuminating light beam  3  and emit light in a characteristic region of the spectrum peculiar to them. This light proceeding from specimen  15  defines a detected light beam  17 . The latter travels through microscope optical system  13  and scanning optical system  12  and via scanning module  7  to deflection means  5 , passes through the latter, and arrives via a detection pinhole  18  at at least one detector  19 , which is embodied as a photomultiplier. It is clear to one skilled in the art that other detection components, for example diodes, diode arrays, photomultiplier arrays, CCD chips, or CMOS image sensors, can also be used. Detected light beam  17  proceeding from and defined by specimen  15  is depicted in  FIG. 1  as a dashed line. Electrical detected signals proportional to the power level of the light proceeding from specimen  15  are generated in detector  19 . Because, as already mentioned above, light of more than one wavelength is emitted from specimen  15 , it is advisable to insert in front of the at least one detector  19  a selection means for the spectrum proceeding from the sample. In the embodiment shown here, the selection means is an SP module  20 . SP module  20  is embodied in such a way that it can acquire a complete lambda scan, i.e. all the wavelengths proceeding from specimen  15  are recorded. The data generated by detector  19  are forwarded to a computer system  23 . At least one peripheral device  27  is associated with computer system  23 . Peripheral device  27  can be, for example, a display on which the user receives instructions for setting the scanning microscope or can view the current setup and also the image data in graphical form. Also associated with computer system  23  is an input means that comprises, for example, a keyboard  28 , an adjusting apparatus  29  for the components of the microscope system, and a mouse  30 . Detected light beam  17  is spatially spectrally divided with a prism  31 . A further possibility for spectral division is the use of a reflection or transmission grating. The spectrally divided light fan  32  is focused with focusing optical system  33 , and then strikes a mirror stop arrangement  34 ,  35 . Mirror stop arrangement  34 ,  35 , the means for spectral spatial division, focusing optical system  33 , and detectors  36  and  37  are together referred to as the SP module (or multi-band detector)  20 . 
     FIG. 2  schematically depicts the arrangement of several device components for FCS analysis in scanning microscope  100 . A conventional scanning microscope  100 , with which a conventional SP module  20  is associated, is used for the FCS analysis. Scanning microscope  100  is equipped with a deflection means  5  that functions as the main beam splitter and is embodied as a beam splitter slider or AOBS™. All available visible laser lines can be coupled in via a first input  40  as light sources. Incoupling of UV radiation or IR radiation, which is usable for multi-photon applications (and also FCS), is accomplished via a second input  41 . In  FIG. 2 , the beam direction toward SP module  20  is indicated by the dashed arrow. The detected light (light proceeding from specimen  15 ) passes through microscope optical system (not depicted in  FIG. 2 ), scanning mirror  9 , deflection means  5 , detection pinhole  18 , optionally a filter wheel  21  to block out the excitation light, and an adjustable beam splitter slider  43 , which contains a mirror for complete deflection into SP module  20 , a beam splitter or a single substrate for partial deflection into SP module  20 , or an empty space. The beam of detected light is collimated by a downstream lens  44 . Located behind this lens  44  is an output  45  out of the stand of scanning microscope  100 . Mounted above this output  45  is a housing  46  in which a changeable filter cube  47  is located. At (removable) beam splitter  48  that is provided in filter cube  47 , the detected light is split into two components, e.g. into a “red” component  50   a  and a “blue” component  50   b . Additional filters  49  provide further blocking of the excitation light and of other undesired light components. These are optional when an AOBS is used as deflection means  5 . One sub-beam, for example “red” component  50   a , is then focused with a lens  51  directly onto first detection channel  52 . The second sub-beam, for example “blue” component  50   b , is focused via an additional deflection mirror  53 , likewise with a lens  51 , onto a second detection channel  55 . Each channel is made up of a standard FC socket  54 , a standard multimode fiber  56  having a light-tight sheath and equipped with an FC connector at each of the two ends, a further FC socket  54 , and an avalanche photodiode (APD)  57 . APDs  57  are characterized by their particularly high detection efficiency as compared with photomultiplier tubes, especially in the visible wavelength region. The two APDs  57  are accommodated together in one housing  58 , to ensure sufficient cooling and light-tightness for the detectors. It is self-evident to one skilled in the art that the avalanche photodiodes can also be replaced in the form of suitable photomultiplier tubes (PMTs), CCDs, etc. that are suitable for photon counting and have a sufficiently high quantum yield. The detectors for FCS can also be mounted directly on housing  46 . The signals of APDs  57  are then conveyed into a computer  60  that is responsible for the FCS analysis. A detector board  61  is provided in computer  60 . Connected to computer  60  is an additional monitor  62  on which the measurement results can be displayed in a wide variety of ways. As already described in  FIG. 1 , scanning microscope  100  has an “independent” computer  23  that is likewise equipped with an independent peripheral device  27 . The peripheral device encompasses, for example, two monitors that belong to scanning microscope  100  as standard equipment. All the control of scanning module  7  and scanning mirrors  9  provided therein, and detection using SP module  20 , is executed via computer system  23 . 
   The method proceeds, in general, in such a way that firstly a confocal overview image of specimen  15  is acquired. One or more points of interest in the image are then identified, e.g. by marking with the cursor, and the illuminating light beam or beams is/are parked there. Beam splitter slider  43  directly in front of output  45  is then set to the empty space or the single substrate. The single substrate has the advantage that one can switch over more quickly between confocal image acquisition and FCS, since no mirror sliders need to be moved between images. FCS imaging and SP module  20  can then be used more or less simultaneously. The fluorescent light is then focused onto the two detection channels  52  and  55 . The APDs provide photon counting. The signal is then conveyed to detector board  61 . An evaluation of the signal (including calculation of the autocorrelation and cross-correlation) is then performed using a software program, and the result is displayed on additional monitor  62 . This method is typically used to determine diffusion rates, concentrations, chemical bonds, etc. 
   A device  65  for generating two foci  72  shifted slightly with respect to one another is arranged between scanning module  7  and deflection means  5 . 
     FIG. 3   a  schematically depicts device  65  for bifocal illumination of a sample for FCS analysis. Illuminating light beam  3  is depicted as a solid line, illuminating light beam  3  is directed, as collimated laser light, onto a dichroic beam splitter  67 . Dichroic beam splitter  67  is embodied, for example, as a beam splitter cube that splits illuminating light beam  3  into a first and a second illuminating light beam  3   a  and  3   b . First illuminating light beam  3   a  or second illuminating light beam  3   b  is tilted relative to the respective other illuminating light beam. According to a preferred embodiment, first illuminating light beam  3   a  travels through dichroic beam splitter  67  in the propagation direction of illuminating light beam  3 . Second illuminating light beam  3   b  is coupled by dichroic beam splitter  67  out of the propagation direction of illuminating light beam  3 . The tilt of first illuminating light beam  3   a  relative to second illuminating light beam  3   b  can be accomplished by way of a rotatable and/or displaceable deflection mirror  68 . The displacement and/or rotation of deflection mirror  68  is accomplished by means of a piezoelement or a galvanometer. It is important in this context that a rotation point  70  of the tilt of deflection mirror  68  lie in the rear focal plane of scanning optical system  12 , and coincide with a rotation axis  71  of scanning mirror  9 . As depicted in  FIGS. 3   b  and  3   c , first illuminating light beam  3   a  and second illuminating light beam  3   b  are imaged into two foci  72  in one plane  75  of specimen  15 . Fluorescent light from the two foci  72  is conveyed in a first and second detection light beam  17   a  and  17   d  back to device  65 , and there combined again. Combination is effected by way of the tilt of deflection mirror  68  and via dichroic beam splitter  67 . In device  65 , a beam trap  69  is associated with dichroic beam splitter  67  in such a way that is arranged opposite deflection mirror  68 . Beam trap  69  serves to eliminate that detected light which is deflected by dichroic beam splitter  67  out of detected light beam  17 . Behind dichroic beam splitter  67 , the combined detected light beam  17  arrives at a lens  73  and is focused by the latter onto confocal detection pinhole  18 . A detector  19  is provided behind detection pinhole  18 . The two foci  72  are arranged in one plane  75  in specimen  15 . Foci  72  themselves have a focal volume in the form of an ellipsoid. Ideally, the focal volumes are smaller in their extent along the Z axis than thickness  78  of specimen  15 . The two foci  72  are arranged in specimen  15  at a spacing  74  from one another. Spacing  74  of the foci is typically greater than diameter  76  of the foci in plane  75 . Modifying the tilt of deflection mirror  68  varies the spacing of foci  72 . If a water-immersion objective of N A =1.2 is used, the result is an extent in the axial or Z direction of approx. 3×200 nm, and a radius in plane  75  of 200 nm. Device  65  defines a rotation axis  77  that coincides with the direction defined by illuminating light beam  3  and detected light beam  17 . Rotation of unit  65  causes dichroic beam splitter  67  and deflection mirror  68  to rotate correspondingly. As depicted in  FIG. 3   b  , foci  72  are rotated relative to one another in plane  75  of specimen  15 . The relative rotation of foci  72  with respect to the X axis is defined by angle α. Angle α lies between line  79  connecting the foci, and the direction of particle flow ν. 
     FIG. 4   a  discloses an embodiment for generating more than two foci  72  in one plane  75  in the specimen. For this purpose, a second device  66  is associated with first device  65 . Only the paths of illuminating light beams  3 ,  3   a ,  3   b , and  3   c  are depicted here. The paths of detected light beams are analogous to the depiction in  FIG. 3   a . With the embodiment depicted here, three foci  72  (see  FIGS. 4   b  and  4   c ) are generated in specimen  15 , all arranged in one plane  75 . Similarly to the description relating to  FIG. 3   a , a first and a second illuminating light beam  3   a  and  3   b  are generated from illuminating light beam  3  using device  65 . A semitransparent beam splitter  80  is provided in first illuminating light beam  3   a . First illuminating light beam  3   a  passes unhindered through the beam splitter. In addition, a third illuminating light beam  3   c  is generated by beam splitter  80 . Third illuminating light beam  3   c  is coupled by means of a further deflection mirror  81  into second device  66 . Beam splitter  80  is arranged in such a way that third illuminating light beam  3   c  is coupled out of the plane that is spanned by first and second illuminating light beams  3   a  and  3   b . In second device  66 , third illuminating light beam  3   c  once again strikes a dichroic beam splitter  67 . If no more than three foci  72  are to be generated in specimen  15 , dichroic beam splitter  67  is embodied as a deflection mirror. As with device  65  in  FIG. 3   a , first device  65  and second device  66  are each equipped with a rotatable and/or displaceable deflection mirror  68 . In each of devices  65  and  66 , a respective beam trap  69  is once again associated with dichroic beam splitter  67  in such a way that it is arranged opposite deflection mirror  68 . Beam trap  69  serves to eliminate that detected light which is coupled by dichroic beam splitter  67  out of detected light beam  17 . 
   The arrangement of focal volumes or foci  72  in specimen  15  is depicted in plan view in  FIG. 4   b .  FIG. 4   c  shows the arrangement of focal volumes or foci  72  in specimen  15 , a cross section through the sample or specimen  15  being depicted. Similarly to the arrangement of foci  72  in specimen  15  depicted in  FIGS. 3   b  and  3   c , foci  72  in  FIGS. 4   b  and  4   c  are likewise arranged in one plane  75 . Foci  72  form, in this context, the vertices of a triangle  85 . Foci  72  in plane  75  of specimen  15  can be modified in terms of their spacing  74  from one another by tilting and displacing deflection mirror  68 . 
   In  FIG. 5 , the detected signals obtained from first detection channel  52  and second detection channel  55  are depicted in the form of correlation functions for the extreme cases of 0° (diamonds  90 ) and 90° (squares  91 ) and the difference (triangles  92 ), for an autocorrelated diffusion correlation time of 1 msec. The autocorrelation function of the detector signal is made up of the autocorrelation functions of the two foci  72  and the two cross-correlation functions of foci  72 , and is ascertained using the following equation:
 
 G (τ)=[ q   4 +(1 −q ) 4   ]G   ac (τ)+[ q   2 (1 −q ) 2   ]G   +   cc (τ)+[ q   2 (1 −q ) 2 ]   G   −   cc (τ).
 
   The individual terms are described in Dittrich et al. (2002), Anal. Chem. 74:4472; G −   cc  is G cc  in this publication, and for G −   cc , angle α is to be replaced by α+180°. This allows calculation of the autocorrelation functions for various orientations (angles) between the double focus and the particle flow. The example in  FIG. 5  shows a case that already leads to a detectable effect in comparison with the diffusion of equal-speed, directed transport. A cross-correlated flow correlation time in of 2 msec, and a spacing of foci  72  equal to twice 1/e 2  of the radii, were selected. The reflectivity of dichroic beam splitter  67  is 50%. The particle velocity as a result of diffusion can be approximately calculated from a maximum  95 , from difference  92 , or from the difference function and the spacing of foci  72 . 
   The correlation function is identical to 0° and 180° for the case of non-symmetric branching as well, meaning that dichroic beam splitter  67  does not split illuminating light beam  3  at the 50:50 ratio. A determination of the sign of the particle flow is not possible. This can be circumvented by calculating higher-order correlation functions, in which symmetry no longer exists in the calculation and there is a difference between 0° and 180°. 
   The invention has been described with reference to a particular exemplary embodiment. It is self-evident, however, that changes and modifications can be made without thereby leaving the range of protection of the claims below.