Patent Abstract:
The invention relates to a high-resolution microscope and to a method for determining the two- or three-dimensional positions of objects. The microscope and method includes the following: (a) The vertical (Z) position of imaged particles or molecules being determined from the orientation and shape thereof by means of an anamorphic lens, preferably a cylindrical lens, in the imaging, (b) the detection beam path being split into at least two partial detection beam paths having different optical path lengths, which are detected at an offset on a detector, (c) activation or switchover being performed by means of a multi-photon excitation process, preferably two-photon excitation. The following are also included: (d) a point-scanning activation or switchover, (e) a line-scanning activation or switchover, (f) the sample is excited and the sample light is detected in the wide-field mode, (g) manually or automatically predetermined sample regions are activated or switched over, (h) the activation or switchover is performed by means of AOTF or SLM or DMD, (i) laser pulses for activating or switching are spectrally split by means of a spectrally splitting element, preferably a grating, (j) an SLM or DMD in the beam path after the grating performs a controlled selection of split laser pulse fractions, (k) the laser wide-field excitation is guided by SLM or DMD, (l) ROIs are selected by SLM or DMD, (m) a multi-photon switching or activation is performed by means of a microlens array, preferably a cylindrical lens array, n) switching and/or excitation is performed by means of a line scanner, and (o) a line detection is performed by means of a spatially resolved sensor, wherein at least two sensor rows, each comprising a plurality of sensors, are illuminated with sample light by means of a slit diaphragm position.

Full Description:
RELATED APPLICATIONS 
       [0001]    The present application is a U.S. National Stage application of International PCT Application No. PCT/EP2010/007595 filed on Dec. 14, 2010 which claims priority benefit of German Application No. DE 10 2009 060 793.5 filed on Dec. 22, 2009, the contents of each are incorporated by reference in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates generally to the field of microscopy and more particularly to high-resolution microscopy. 
       BACKGROUND OF THE INVENTION 
       [0003]    In principle, the optical resolution of a light microscope, such as a LSM, is limited with regard to diffraction by the laws of physics. For an optimal resolution within these limits particularly illumination configurations are known, such as 4Pi arrangements or arrangements with fields of stationary waves. Here, the resolution can be considerably improved, particularly in the axial direction, in reference to classic LSM. 
         [0004]    Using non-linear depopulation processes, the resolution can be further increased in reference to a diffraction-limited confocal LSM. Such a method is described, e.g., in U.S. Pat. No. 5,866,911. Several approaches are known for the depopulation process, such as described in DE 4416558 C2, U.S. Pat. No. 6,633,432, or 10325460 A1. 
         [0005]    In recent years, various methods to overcome the diffraction limit have been developed and applied in fluorescence microscopy (PALM structured illumination WO 2006127692; EP 1157297 B1). 
         [0006]    A presently particularly advantageous method developed for high-resolution fluorescence microscopy is based on the highly-precise localization of individual molecules. It is known that the localization, thus the determination of the position of an individual fluorescent molecule, is not subject to limits of diffraction. 
         [0007]    This localization can occur with highly sensitive cameras in the wide field with a precision up to the nm-range, when sufficient protons of the molecule can be detected. In high-resolution microscopy based on localization an image is composed from the molecule positions obtained in this manner. Here it is critical that at any given time only one subset of molecules of the sample are in a fluorescent state so that on average the “closest neighbor” distance of the active molecules is always greater than the PSF (point spread function) of the microscope. This is achieved by using optically or chemically switched fluorophores: in a tightly marked area of a sample, by way of irradiation of suitable conversion wavelength, stochastic subsets of fluorophores are switched in the examined area into the fluorescent state. Here, the density of the spot is adjusted such that a continuous localization of the positions of the molecules is permitted. This optic switching method is used, for example in PhotoActivated Localization Microscopy (PALM). This fundamental method is described in the literature (listed hereinbelow) in detail, using different variations, see literature items [1-6]. 
         [0008]    Here, the high-resolution methods (PALM, STORM, D-STORM etc.) primarily differ in the selection of the fluorophores and the type of optic switching process. 
         [0009]    However, all methods have in common that the localization of the molecules occurs by an imaging process on a highly sensitive camera (e.g., EMCCD). The quasi-punctual light source (molecule) is here displayed by the point spread function (PSF) of the microscope over several camera pixels. The precise position of the molecule in the x/y-level can now be determined either by fitting the known PSF (Gauss) or by a determination of the focus or by a mixture of both (Gaussian mask) (see the literature, citing different algorithms). 
         [0010]    Typical precisions of localization range (depending on the experimental conditions) from 5 to 30 nm; this then also represents approximately the lateral resolution of this method. Practically, this calls on the one hand for molecules not being located too close to each other and on the other hand for an illustration of the examined structures as completely as possible, so that many individual images (typically 10,000-20,000) must be taken of the sample. This leads to a rather extensive imaging period as well as to problems with regard to the adjustment of the switching intensity, particularly when the sample and/or the structures of interest are marked very inhomogeneously: in order to prevent losing any information the switching intensity must always be adjusted to the area of the sample marked most densely. 
         [0011]    In general, the above-described method based on localized high resolution is limited to surfaces and/or 2 dimensions, because the localization of the individual colorant molecules are considerably more complex in the third dimension (z-direction). The number of accepting individual molecules for illustrating the structures increases accordingly in the three-dimensional case. 
         [0012]    Another problem for the three-dimensional high-resolution display in the depth of a sample lies in the limited penetration depth: as already known from the classical fluorescence microscopy and laser-scanning microscopy, the increasingly dispersed excited radiation in the depth of the sample leads to an increase of the background signals with a simultaneous reduction of the actual wanted signal. 
         [0013]    In addition to the undesired photo bleaching of sample sections outside the focused level, it also occurs in the PAL-M method that in the depth of the sample undesired switching of the fluorophores occurs by the activating radiation in the layers not presently measured. 
         [0014]    In prior art there is the general need for a high-resolution display of fluorescence in three dimensions with high penetration depth and “sectioning” (thus measuring a layer and here avoiding exciting/bleaching and particularly switching the photo-convertible fluorophores in the layers located above or below) and an increase in recording speed. 
       SUMMARY OF THE INVENTION 
       [0015]    According to the invention, it has been recognized that a three-dimensional microscope with increased resolution can be realized using the synergies of the following advantageous technologies and arrangements in the arrangements and methods described in detail in the following: 
       High-resolution Determination of an Axial (Z-) Position of Molecules: 
     Astigmatism/cylinder Lens ([ 9 ]) 
       [0016]    In this approach a weak cylinder lens is inserted into the detection radiation path, which leads to an astigmatic PSF. Accordingly, the image of the molecule is elliptically distorted when the molecule is located above or below the symmetry point of the PSF. Information can then be extracted concerning the z-position of the molecule from the orientation and extent of said distortion. The anamorphotic optic (cylinder lens) is advantageously used according to the invention for determining the vertical position of the molecule by detecting the form and/or size and realized in a microscope using the following methods and arrangements. 
       Detection in Two Levels (Biplanar Detection) (Toprak et al., Bewersdorf et al. [7,8]) 
       [0017]    Here, a 50/50 beam splitter is inserted into the detection radiation path, splitting the image into two partial images (duplicated). These two images are either displayed on two identical cameras or side-by-side on a camera chip. An optic difference of path lengths is introduced into one of the two partial radiation paths such that two object levels develop from the two partial radiation paths, apart from each other in the z-direction, by approximately half to one z-PSF (700 nm). The z-position for molecules located between these two levels can now be determined, e.g., by way of subtraction of the two partial images of the molecule and/or by fitting a three-dimensional PSF or a similar algorithm. 
         [0018]    The splitting of the detection radiation path and an off-set detection of the split radiation paths, showing a difference in length, as well as the other methods and applications according to the invention are implemented in a microscope. 
       Penetration Depth: Multiphoton Microscopy 
       [0019]    Prior art of multi-proton microscopy, particularly 2-photon (2P) excitation and its advantages, are used in a targeted fashion for the methods and arrangements according to the invention described in the following. 
         [0000]    The 2P Switching of Colorants that can be Converted 
         [0020]    The possibility of a 2P excitation applies not only for the typical fluorescence excitation of known colorants but in principle also for the change of status of so-called switchable fluorophores or photo-switches. This represents fluorophores, which by the irradiation of a switching wavelength can be set into a fluorescent or non-fluorescent state, depending on the initial state (cf. PALM, STORM, etc.). Here, the switching wavelength may also be equivalent to the excitation wavelength. Such photo switches are essential for the above-mentioned high-resolution methods. 
         [0021]    In various publications, see literature item [10], it was reported that some switchable fluorophores can also be switched with a 2-photon excitation, e.g., the proteins dronpa, eosFP, kaede, kikume, PA-GFP (references 2P switching). 
         [0022]    The 2P activation (switching into a state that can be activated) is particularly used in the point scanning mode, but also in the line scanning mode, preferably with wide-field fluorescence excitation and wide-field detection, individually or with other arrangements and methods used according to the invention. 
         [0023]    They are particularly beneficial in connection with the marking of pre-determined regions (ROI), which, for example, are detected and marked in a preliminary image, such as certain cells or other interesting biological fields, particularly by: 
         [0024]    AOTF control, SLM control, or DMD control of the impingement with switching radiation and/or excitation radiation. 
         [0025]    This occurs two-dimensionally and three-dimensionally (in the image stack), also in connection with the other methods and arrangements in a microscope listed according to the invention. 
       Temporal Focusing of the Switching Radiation 
       [0026]    Using the so-called temporal focusing, the effect of the depth discrimination, which develops in the point-scanning 2P microscopy by the square intensity dependency of the excitation in combination with strong focusing, a wide-field display can also be achieved. For this purpose, e.g., in literature item [11] short laser pulses are split spectrally via a grid; this grid is then displayed via the lens of the microscope. This leads to the different spectral components of the pulse assuming different optic paths and only merging back in the focal area in order to here form the original short laser pulse. Here, the highest power of the pulse is only maximal in the focal area, which in the context of the above-mentioned square intensity dependency of the 2P excitation leads to a depth discrimination, but now at a wide field. 
         [0027]    This may be connected by SLM or DMD control with the ROI function, as mentioned above. 
       2P Multi-spot Imaging 
       [0028]    with a grid micro-lens array or rotary micro-lens disk, e.g., in literature item [14] and references therein is realized in a microscope with other methods and arrangements listed. 
       Switching and Excitation Via a Lens Scanner 
       [0029]    are realized in a microscope with the other methods and arrangements listed. 
         [0030]    Contrary to the arrangements and methods described in the following, prior art could not achieve the synergies and advantages according to the invention:
       PALM and similar methods yield a two-dimensional high-resolution fluorescent image, but only at the cover-glass boundary under TIRF excitation   Using bi-planar or astigmatism charges additionally the z-resolution can be increased; the measuring of thicker samples is particularly problematic such that only layers of a dimension of one PSF can be measured. Layers above or below this can be sequentially measured as an image stack, as is common in microscopy; however, since this represents (laser) wide-range excitation and switching, the layers above and below are also switched and/or bleached as early as during the measuring of the present layer, and thus they cannot be measured or only to a limited extent.   Temporal focusing for switching the fluorophores literature item [12] was demonstrated and a 2P-comparable sectioning was observed. However, this way no real 3D-high resolution can be achieved.   2P switching by a point-scanned cw laser in connection with wide-field detection was demonstrated in item [13]. Here, too, it is sectioned; however, no 3D-resolution is possible.   In all localization-based high resolution methods based on photo-switches presently the intensity of the switching laser per image must be adjusted to the highest marker density inside the observed sample range.       
 
       LITERATURE 
       [0000]    
       
         [1] Betzig et al. Science 313, 1642-1645 (2006) 
         [2] Hess et al., PNAS 104, 17370-17375 (2007) 
         [3] Hess et al., Biophys J. 91, 4258-427 (2006) 
         [4] Schroff et al., PNAS 104, 20308-2031 (2007) 
         [5] Rust et al., Nat Methods 3, 793-796 (2006) 
         [6] Egner et al., Biophys J. 93, 3285-3290 (2007) 
         [7] Toprak et al., Nano Lett. 7, 2043-2045 (2007) 
         [8] Juette et al., Nature Methods 5, 527 (2008) 
         [9] Huang et al., Science 319, 810 (2008) 
         [10] Marriot et al. PNAS Vol. 105, #46, pg 17789, 2008, Ivanchenko et al. Biophys J, vol. 92, pgs 4491-4457, 2007, Watanabe et al., Opt Exp., vol. 15, pg 2490, 2007, Schneider et al. BioPhys J, vol. 89, pg 1346-1352, 2005 
         [11] Oron et al, Optics Express 13, 1468 (2005) 
         [12] Vaziri et al., PNAS 105, 20221 (2008) 
         [13] Folling et al., ChemPhysChem 9, 321, (2008) 
         [14] Pawley, Handbook of Biological Confocal Microscopy (3 rd  Edition) 
         DE 19829981A1: Regions of Interest 
         DE 19930532A1: SLM 
         DE10259443 A1: Pulse combination in the sample 
         DE 19835072A1: DMD 
         U.S. Pat. No. 5,867,604: Structured illumination 
       
     
         [0055]    The invention further has features of the independent claims. 
         [0056]    It is realized in a high-resolution microscope and a method for the two- or three-dimensional determination of the position of objects, particularly individual fluorophores, preferably for the spatially high-resolution fluorescent microscopy of a sample marked with marking molecules, which can be activated or switched by a signal such that only in the activated or switched state they can be excited to emit fluorescent radiation, with the method comprising the following steps:
       1) Introducing the signal to the sample such that only a partial amount of the marker molecules present in the sample are activated, with partial sections developing in the sample in which activated marker molecules show a distance from the closest neighboring activated marker molecules greater than or equivalent to a length, which results from a predetermined optic resolution,   2) excitation of the activated molecule to emit fluorescent radiation,   3) detection of a fluorescent radiation with a predetermined optic resolution, and   4) generation of an individual image from the luminescent radiation recorded in step 3), with the geometric locations of the marker molecules emitting the fluorescent radiation being determined with a local resolution enhanced beyond the predetermined optic resolution, with the steps being repeated several times and the multitude of individual images yielded in this manner being combined to an overall image.       
 
         [0061]    The invention further advantageously comprises at least one of the arrangements a) to q) of claim  1 . 
         [0062]    The invention furthermore comprises advantageously at least one of the processing steps a)-o) of claim  2 . 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0063]    In the following, the invention is explained in greater detail using the schematic drawings, in which: 
           [0064]      FIG. 1  is a schematic illustration of an activated marker molecule in a volume with limited resolution; 
           [0065]      FIG. 2  is a schematic illustration of the image of various activated and non-activated marker molecules on a locally resolving detector; 
           [0066]      FIG. 3  is a flow chart for the image generation in the PALM method; 
           [0067]      FIG. 4  is explanatory illustrations allocated to the flow chart of  FIG. 3  of the marker molecules displayed on the detector of  FIG. 2 ; 
           [0068]      FIG. 5  is schematic illustration of a microscope concerning PAL-microscopy; 
           [0069]      FIG. 6  schematically shows a depth-selective 3D-high-resolution fluorescence microscope (schematically) in a 2-channel embodiment; 
           [0070]      FIG. 7(   a ) shows an adjustment of the switching intensity; 
           [0071]      FIG. 7(   b ) shows a schematic image stack in the x-z cross section; 
           [0072]      FIG. 8  schematically shows a depth-selective 3D high-resolution microscope; 
           [0073]      FIG. 8(   a ) shows the radiation beam from the light source to the sample in an enlarged fashion; 
           [0074]      FIG. 9  schematically shows a depth selected high resolution microscope with a scanning micro-lens dish; 
           [0075]      FIG. 10(   a ) shows a principle of the localization-based confocal high-resolution microscopy with a line sensor; 
           [0076]      FIG. 10(   b ) shows a sensor with an engaged pixel structure; 
           [0077]      FIG. 10(   c ) shows an example to realize an engaged pixel structure with existing pixel geometries by way of masking; 
           [0078]      FIG. 11  shows schematically a potential embodiment of a depth-selective high-resolution microscope based on the sensor principle; 
           [0079]      FIG. 12  shows a wide-field radiation path with a widened light source and a spatially resolving area detector, for example a CCD camera; 
           [0080]      FIG. 12(   a ) shows an enlarged detail of the variable image splitter module BM according to the invention; 
           [0081]      FIG. 12(   b ) shows, in an enlarged view, point images of molecules  1 ,  2 ,  3 ; 
           [0082]      FIG. 13  shows an enlargement of four images of four sample levels; 
           [0083]      FIG. 13(   a ) shows another advantageous embodiment of a module for splitting a camera image into 4 partial images of identical intensity for z-high resolution; 
           [0084]      FIG. 13(   b ) shows a monolithic embodiment with 3 beam splitter cubes; and 
           [0085]      FIG. 14  shows an advantageous combination of an embodiment according to  FIG. 13  with displaceable prisms according to  FIG. 12 . 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0086]      FIG. 1  shows schematically a marker molecule  1 , which has been excited for fluorescence. Of course, the detection of fluorescence requires a plurality of excitations, because each excitation yields precisely one fluorescent proton and the detection of radiation requires an integration of many fluorescent protons. The fluorescent radiation emitted by the marker molecules  1  can be detected in a microscope based on physical principles only in a limited optic resolution. Even if the microscope reaches the diffraction limit of the optic resolution the photons of the fluorescent marker molecule  1  are still distributed due to diffraction and thus detected in a diffraction disk  2 . The microscope therefore displays an object principally larger than the geometric extension of the marker module  1 , drawn in  FIG. 1  schematically as a black circle, which is illustrated in  FIG. 1  by the diffraction disk  2 . The size of the diffraction disk  2  depends on the quality of the microscopy device used and is defined by the half-width of the point spread function of the optic display. Actually, this represents not a two-dimensional object but a diffraction volume, which the fluorescent photons penetrate. In the two-dimensional illustration of  FIG. 1  this appears as a disk, though. The term diffraction disk is here used generally for a maximum resolution volume, which the optic used can achieve. It is not mandatory for the optic used to work at the diffraction limit, even if this is preferred. 
         [0087]    In order to allow more precisely localizing the marker molecule  1  within the diffraction disk  2  the PALM method is used, already described above in a general fashion. It activates individual marker molecules, with, in this description, generally the term activation relates to the activation of certain luminescent features of the marker molecules, thus both switching on the luminescent excitation as well as a change of the spectrum of luminescence emitted, which is equivalent to switching on certain luminescent features. In the exemplary embodiment described here the activation is caused by optic activation radiation. However, different, non-optic activation mechanisms are also possible. 
         [0088]    The activation now occurs such that there are at least some activated molecules, with their focal point not being within the diffraction disk of other activated molecules, i.e. which can still be distinguished, at least within the optic resolution. 
         [0089]      FIG. 2  shows schematically an exemplary situation on a detector  5 , which integrates the protons in a spatially resolving fashion. As discernible, there are areas  3 , in which the diffraction disks of adjacent marking molecules overlap. Here, as is discernible in the left area  3  of  FIG. 2 , only those marker molecules are relevant which have previously been activated. Non-activated marker molecules  1 ′ fail to emit the determined fluorescent radiation, which is detected on the matrix detector  5 ; thus they are irrelevant. 
         [0090]    In the areas  4 , e.g., areas  4  located in the center of the matrix detector  5 , the marker molecules  1  are located such that their diffraction disk  2  overlaps with none of the diffraction disks of other activated marker molecules  1 . The right area of the matrix detector  5  shows that areas  3 , in which diffraction disks of activated marker molecules overlap, may indeed be located adjacent to areas  4  in which this is not the case. The right area  4  illustrates additionally that the neighborhood of an activated marker molecule  1  is irrelevant for the detection of a non-activated marker molecule  1 ′, because such a marker molecule  1 ′ emits no fluorescent radiation detected by the matrix detector  5 ; thus it is not fluorescent. 
         [0091]    In order to record a detailed image beyond the optic resolution predetermined by the device, with the image being a high-resolution image in the sense of this description, now the steps schematically illustrated in  FIG. 3  are used. 
         [0092]    In a first step S 1 , using a switching signal, a subset of the marker molecules is activated; they are therefore switched from a first state, in which they cannot be excited to emit the certain fluorescent radiation, into a second state, in which they can be excited to emit the certain fluorescent radiation. Of course, the activation signal can also lead to a selective deactivation, thus in step S 1  an inverse process may be used, too. It is essential that after the step S 1  only a subset of the marker molecules can be excited to emit certain fluorescent radiation. The activation and/or deactivation (in the following for reasons of simplification only the case of activation is being discussed) occurs independent from the marker molecules used. In a colorant, such as DRONPA, PA-GFP, or reversibly switchable synthetic colorants (such as Alexa/cyan constructs), the activation occurs by optic radiation; the switching signal is therefore a switching radiation. 
         [0093]      FIG. 4 , shown under  FIG. 3 , illustrates in detail the condition after step S 1 . Only a subset of the marker module  1 _n is activated, here. The marker molecule of this subset is represented by a filled black dot. The remaining marker molecules have not been activated in this step. They are marked  1 _n+1 in detail a of  FIG. 4 . 
         [0094]    Marker molecules, which have been activated, may then be excited in a second step S 2  to emit fluorescent radiation. Used as fluorescent colorants are preferably fluorescent proteins, known from prior art, such as PA-GFP or also DRONPA. The activation occurs in such molecules with radiation at a range of approx. 405 nm, the excitation for fluorescent radiation at a wavelength of approx. 488 nm, and the fluorescent radiation is in a range above 490 nm. 
         [0095]    In a third step S 3  the emitted fluorescent radiation is detected, for example by integration of the recorded fluorescence photons, so that the situations develop on the matrix detector  5  shown in the detail b of  FIG. 4  located thereunder. As discernible, the diffraction disks of the activated marker molecules  1 _n are not overlapping. The size of the diffraction disks is determined by the optic resolution of the display on the matrix detector  5 . Additionally, in the detail b of  FIG. 4  (theoretic) diffraction disks of fluorescent molecules are drawn, not included in the non-activated group  1 _n+1. Due to the fact that these non-activated marker molecules emit no fluorescent radiation no fluorescent radiation located in the (theoretic) diffraction disks can compromise the detection of the fluorescent radiation of the subset  1 _n of the activated marker molecules. 
         [0096]    In order for the subset  1 _n to overlap as few diffraction disks as possible such that the marker molecules cannot be distinguished any longer, the activation energy shall be adjusted such that the subset  1 _n represents only a relatively small portion of the overall number of marker molecules so that statistically many marker molecules can be distinguished in reference to a volume that can be dissolved by the optic arrangement. 
         [0097]    In a fourth step S 4  the location of the fluorescent marker molecules is determined by way of calculation from the diffraction distribution of the fluorescent disks, by which the resolution disclosing the position of the marker molecules that can be activated is sharpened beyond the resolution of the optic arrangement, as shown in the detail c of  FIG. 4 . 
         [0098]    Alternatively to the calculated determination it is generally possible to enhance the recorded fluorescent radiation in a non-linear fashion and this way to enhance the resolution beyond the optic arrangement with lesser expense. The non-linear enhancement may be performed, for example, according to the function S=A*F N  (equation 1) or S=A*exp F/w  (with w=10 −N  (equation 2)), with F representing the amplitude of the fluorescent signal, A a norming factor, and N an integer greater than 1. A strong non-linear dependency of the parameter S from F is particularly advantageous, thus, e.g., high values for N in the equations 1 or 2. Of course, other functions may also be selected. In general, the non-linearity is preferably selected such that the half-width of the diffraction disk is equivalent to a desired spatial resolution for the determination of location of the marker molecules. In addition to a non-linear enhancement, a non-linear damping may also be used. Here, fluorescent signals with low amplitudes or intensities are damped, while strong signals remain at least largely undamped. Of course, any combination of non-linear enhancement and damping may also be used. 
         [0099]    A fifth step S 5  combines the marker molecules, with their statement of location being determined more precisely, to form an individual image, with its spatial resolution being enhanced beyond the optic resolution. However, it only includes information regarding the previously activated subset of the marker molecules. 
         [0100]    In a sixth step S 6  the individual image is inserted in a manner known per se into an overall image. Subsequently it is returned to step S 1 , with the previously fluorescent molecules now having to be deactivated. A deactivation may occur depending on the marker molecule by a separate radiation or by the activated state fading. Additionally it is possible to bleach already displayed marker molecules by way of exciting radiation. 
         [0101]    With each run another individual image is yielded, contributing to the overall image. In the next run, another subset of the marker molecule is activated, e.g., the subset  1 _n+1 shown in  FIG. 4 . 
         [0102]    By the repeated run through the steps S 1  through S 6  the overall image of individual pictures of these individual runs is composed, which state the location of the marker molecules with a spatial resolution which is sharper compared to the resolution of optic imaging. By a respective number of iterations this way a high-resolution overall image is generated successively. The reduction of the diffraction disk here occurs in the method preferably in all three spatial dimensions when several image stacks distanced in the z-direction are recorded. Then the overall image comprises the high-resolution local information of the marker molecules in all three spatial directions. 
         [0103]      FIG. 5  shows schematically a microscope  6  for a high-resolution imaging of a sample  7 . The sample is marked, for example, with the colorant DRONPA (cf. WO 2007009812 A1). In order to activate as well as to incite fluorescence the microscope  6  comprises a light source  8 , which has individual lasers  9  and  10 , with their radiation being combined via a beam combiner  11 . The lasers  9  and  10  may, for example, emit radiation at 405 nm (activation radiation) and 488 nm (fluorescent excitation and deactivation radiation). Additionally, colorants are known (e.g., the colorant named DENDRA (cf. Gurskaya et al., Nature Biotech., Volume 24, pages 461-465, 2006)), in which the activation and excitation of fluorescence can occur at the very same wavelength. In this case one laser is sufficient. 
         [0104]    An acoustic-optic filter  12  serves for the selection of wavelengths and for a rapid switching or dimming of individual laser wavelengths. An optic  13  focuses the radiation via a dichroitic beam splitter  14  in an aperture diaphragm of the lens  15  so that the radiation of the light source  8  impinges the sample  7  as a wide-field illumination. 
         [0105]    The fluorescent radiation developing in the sample  7  is collected via the lens  15 . The dichroitic beam splitter  14  is designed such that it allows the fluorescent radiation to pass so that it reaches a tubular lens  17  via a filter  16 , so that overall the fluorescent sample  7  is displayed on the detector  5 . 
         [0106]    In order to control the operation of the microscope  6  a control device is provided, here embodied as a computer  18  with a display  19  and a keyboard  20 . The processing steps S 2  to S 6  occur in said computer  18 . Here, the image rate of the matrix detector is decisive for the overall measuring period so that a matrix detector  5  with an image rate as high as possible is advantageous in order to reduce the measuring period. 
         [0107]    The reference characters in  FIGS. 6-11  represent the following:
       Pr: sample   O: lens   D, D 1 , D 2 , D 3 , D 4 : dichroitic beam splitter   L 1 -L 2 : light sources   Bt 1 , Bt 2 : image splitter module (detection on several levels)   K 1 , K 2 : area receiver (camera)   S Scan module (schematic) with X/Y scanners   Sx: one-dimensional scanner (in the x-direction)   TL: tubular lens   SO: scan lens   SLM: spatial light modulator   G: grid   SML: micro-lens array   SE: single sensor of a sensor arrangement   B 1 , B 2 : adjustable slit diaphragm   Ld 1 , Ld 2 : line detector   ZL: anamorphotic optic, such as a cylinder lens   SF: beam splitter for linear focus (anamorphotic optic)       
 
         [0126]    Most drawings have in common that, similar to the situation described in  FIGS. 6-10  using the example of the PAL-M method, a first laser L 1  may be provided for switching (activating) the colorant and lasers L 2 , L 3  are provided for exciting fluorescence/deactivating colorants in the sample Pr in the wide field and one or more cameras (preferably CCDE) are provided for wide-field detection. 
         [0127]    Included in the disclosure are further processes for a time-related activation/deactivation of colorants to generate high-resolution microscopic images of prior art as described at the onset. 
         [0128]    Multi-level detection is understood as, for example, particularly an image splitter module according to  FIGS. 12-14 , and/or an anamorphotic illustration as well as embodiments of prior art (see literature items [7, 8]). 
         [0129]    Here, “ROI,” regions of interest, are considered regions, automatically or manually preselected for example based on an overview image, which can be impinged selectively with radiation. 
         [0000]    Bi-planar Detection Diagram with 2P Switching Using Point Scanners 
         [0130]      FIG. 6  schematically shows a depth-selective 3D-high-resolution fluorescence microscope in a 2-channel embodiment for 2 different fluorophores, simultaneously observed, comprising two cameras K 1 , K 2 . 
         [0131]    A point-scanning scan module (S) follows, in the direction of radiation, a laser (L 1 ) to switch via a non-linear excitation, particularly 2P excitation, which may be a ps laser diode or even a cw diode laser in the range of 780-830 nm (or another wavelength suitable for the 2P switching process); the laser L 1  is coupled into the radiation path via the dichroit D 1 . 
         [0132]    Lasers for the wide-field excitation L 2  and L 3  in various wavelengths are coupled via D into the radiation path. The detection of fluorescence occurs via D 2  in the transmission in the direction of the cameras K 1 , K 2  with a splitting into two color channels for different wavelengths of two fluorophores via D 3 . For each color a splitting of the image via image splitting modules (Bt 1  and Bt 2 ) can occur as described in  FIG. 12-14 . 
         [0133]    Here, advantageously a fluorescence excitation occurs by laser wide-field illumination as well as wide-field detection by (sensitive) cameras, such as is common in PALM and similar localization-based high-resolution methods. 
         [0134]    The detection occurs here beneficially with a biplanar (multi-level) detection diagram, (see items [7, 8]),  FIGS. 12-14  for the highly precise localization of the molecules, also on the z-axis. 
         [0135]    Here, the image is advantageously (see  FIGS. 12-14 ) split by the image splitting module (Bt 1 / 2  in  FIG. 6 ) such that two partial images develop with half the intensity each, with their conjugated object levels being off-set by, for example, approx. half the axial PSF. 
         [0136]    These partial images are displayed side-by-side on the camera sensor (K 1 /K 2 ) and can be assessed accordingly [7, 8] in order to determine the respective z-position of the individual fluorophores from the 2 partial images. 
         [0137]    A particularly beneficial embodiment of this image splitter module is the object of  FIGS. 12-14  with the following advantages:
       Telecentric   No changes of the display scale as a function of the z-position   Parallel impingement upon the detector (→no z-dependent distortion of PSF)   Adjustable z-splitting and thus ability to adjust to different lenses and embedding media   Ability to adjust the splitting to zero, whereby   The reference image level may be located on the sample surface; the adjustable splitting then occurs into the sample (and not into the cover glass as in embodiments of prior art)   Ability to remove the splitting in order to utilize normal camera images with a full sensor.       
 
         [0145]    It is particularly advantageous for the range of depth of focus achieved by the described image splitter module according to  FIGS. 12-14  to be variably adjusted to a similar range as the minimal layer thickness of the two-photons excitation (for example, 700 nm) and/or as shown in the arrangement according to  FIGS. 12-14 . Here, an optimal overlap develops of an activated (=switched) layer and a layer measured with high-resolution. 
         [0146]    The switching (photo-converting) of the fluorophores from their non-excited into their excited state (condition for PALM) occurs here via a focusing, point-scanning excitation beam, with its wavelength being selected such that the switching process occurs by a 2-photon (or 3-photon, generally multiphoton) absorption process, while the fluorescent excitation and detection of the fluorophores switched in this manner occur in the wide-field. 
         [0147]    Based on the stochastic activation of the photo-switches in the PALM method the grid-based activation is not necessarily synchronized with the imaging of the camera. 
         [0148]    The 2P switching (converting) beneficially leads to the “sectioning” known from the 2-photon microscopy, thus the selective excitation of only the molecules in the focal area. This way a z-resolution can be achieved similar to the confocal microscopy without the use of any confocal pinhole being required. This way the method can easily be combined with the camera-wide-field detection required for PALM. 
         [0149]    Additionally, contrary to the confocal detection, any out-of-focus switching is avoided, which is particularly important for the imaging of high-resolution z-stacks. 
         [0150]    It is known that the photo-conversion (the switching) of the typically used photo-switches for PALM microscopy requires extremely low intensities only. Accordingly the 2P effect can also be achieved with cost-effective cw lasers, beneficial in reference to expensive short-pulse lasers for 2P microscopy. 
         [0151]    For fluorophores or applications and/or sample preparations requiring higher power for the 2P switching process, a laser diode operated in the pico-second mode (exemplary embodiment) may be used or other, cost-effective ps laser systems. 
         [0152]    The typical 2P switching wavelengths for common PALM fluorophores range from 760-850 nm, a range well covered with cost-effective semiconductor lasers. 
         [0153]    In  FIG. 7 , in a), an adjustment occurs of the switching intensity of the point-scanned switching laser to the marking density in the sample. In the area I marked thinner the switching intensity is increased in order to adjust the rate of the localized molecules to the one in the denser marked area II.”  FIG. 7   b  shows a schematic image stack in the x-z cross section. 
         [0154]    By a targeted switching of the scanned activation laser in only two previously manually or automatically defined ROIs the undesired switching above or below the just detected layer is avoided. This is particularly advantageous when recording z-stacks comprising several such layers. 
         [0155]    Using the point-scanned switching beam ROIs (regions of interest—see DE 19829981A1 can be defined, with fluorophores being activated therein; however, in TIRF-PALM all regions of the sample are always activated. This way, the method described here, e.g., the switching intensity within a frame, can be optimally adjusted to the marker density of the sample (cf.  FIG. 7   a )). This way, the imaging period can be reduced, which is important, particularly for taking images of 3D-image stacks. Additionally, the localization rates can also be adjusted to other fields in the sample marked with different fluorophores. 
         [0156]    Another advantage comprises that undesired switching of molecules in sample fields above and below the z-layer most recently to be recorded can be minimized (in addition to the above-mentioned intrinsic sectioning of the 2P excitation), by the activation radiation exclusively being switched on in the sample fields with colored structures ( FIG. 7   b )). 
         [0157]    In principle, both effects can also be achieved with the classical  1 P switching source, when it is focused and scanned. However, the very beneficial sectioning of the 2P absorption is not achieved, here. 
         [0158]    An advantageous method for a sequential process for a numerically controlled feedback for 2P switching can occur according to the invention. 
         [0159]    In inhomogeneously colored samples it allows a quicker achievement of the comparable localization density in different sample fields and thus a faster recording time. 
         [0160]    After the recording of the camera image (optional: real-time localization) a determination occurs of the number or number density of the localized molecules per ROI as well as a comparison {area ROI: number} with the PSF in such a way: 
         [0161]    When {area ROI: number}&gt;&gt;is (larger than) the PSF the intensity of the 2P switching beam for this ROI is increased. 
         [0162]    When {area ROI: number}˜=is (in the area) of PSF, the intensity of the 2P switching beam for this ROI is reduced. 
         [0163]    The issue of the respective intensity modulation to the scanning 2P switching beam follows and a subsequent camera image is recorded. 
       A Method According to the Invention for ROI-Controlled Feedback for 2P Switching: 
       [0164]    minimizes the undesired switching of molecules in the sample fields above and below the just measured sample level by the following steps:
       Defining one or more ROIs automatically or manually based on a first image, recorded with high switching and/or excitation intensity, or based on an image obtained with other contrast functions (DIC)   Issuing the respective intensity modulation on the scanned  2 P switching beam so that the switching radiation is “on” only within a predetermined ROI   Within these predefined ROIs, similar to 9.3.2, the adjustment of the switching intensity can occur to the count rate of the molecules.       
 
         [0168]    The described regulation of the switching intensity is particularly advantageous for samples colored with different fluorophores, which are measured simultaneously. 
         [0169]    For example, perhaps different switching rates of different molecules can be adjusted by the spatial adjustment of the switching intensity and this way the recording period for multi-color measurements can be reduced. The ROIs can be defined based on sample characteristics or can be superimposed over the image as a regular grid with fields of a suitable size; the latter is particularly advantageous for the following explanations with wide-field switching illumination (temporal focusing, spinning micro-lens disk). 
         [0000]    Bi-planar Detection Diagram with 2P Switching Using Line Scanner 
         [0170]    In order to achieve sufficient activation of a stochastic subset of fluorophores per camera frame necessary for PALM, particularly for quick imaging rates, a very high scanning speed of a point scanner is required. The use of a line for the 2P switching accordingly reduces the scanner speed. The reduction of the peak intensity allocated to the line can be tolerated due to the low switching intensities necessary. 
         [0171]    Instead of a point scanner, a line scanner can also be used for 2P excitation in  FIG. 6  (see also  FIG. 11 ) 
         [0172]    The low intensities required for switching colorants also render the use of a scanned line possible in connection with the 2P switching process. 
         [0173]    This line may be created with an anamorphotic optic, for example, and moved with a one-dimensional scanner through the object. 
         [0174]    Here, the advantage lies in the fact that a more rapid scanning is possible, because only 1D-scan is required. 
         [0175]    Due to the fact that the activation occurs stochastically and the detection occurs in a wide field it is not necessary for the line to be synchronized with the imaging process. 
         [0000]    Bi-planar Detection Scheme with 2P Switching Via Temporal Focusing 
         [0176]      FIG. 8  schematically shows a depth-selective 3D high-resolution microscope with L as the activating laser; L 2 , L 2  as the excitation laser, a grid G: grid; a SLM: spatial light modulator (see here for example DE 19930532A1); D 1 -D 4  are dichroitic beam splitters and/or combiners, Bt 1  and  2 : image splitter modules, K 1  and K 2  here represents cameras. 
         [0177]      FIG. 8   a  shows the radiation beam from the light source to the sample in an enlarged fashion. 
         [0178]    The radiation path is shown with a continuous line and the image [thereof] in dot-dash lines. 
         [0179]    This also applies respectively for the illustration of  FIG. 9 . 
         [0180]    The lens arrangement O is shown schematically by a lens. 
         [0181]    The grid is located in an interim image and is displayed in the next interim image (dot-dash lines) in which the SLM is located. This in turn is displayed in the sample. 
         [0182]    However, the radiation bundle (continuous line) of the laser is “off-set”; in the sharp interim images (grid, SLM, sample) the illustration spot is maximally unfocused, here. 
         [0183]    Here, once more a ROI functionality can occur as described above, with the difference that the respective switching intensity is now adjusted via the SLM in the interim image. 
         [0184]    This arrangement advantageously operates without any moving parts (non-scanned) and yields similar flexibility as the above-mentioned scan arrangement. 
         [0185]    The depth selection is now achieved via the 2P switching effect in connection with the temporal focusing [11 and references therein]. For this purpose, using the widened beam of a short-pulse laser L 1  with a wavelength suitable for the 2P excitation of the switching process, a grid G is radiated. The radiated field of the grid is then displayed in the sample. The grid splits the spectral portions of the laser pulse such that the original short pulse form is achieved only in the focus of the display (and perhaps interim images) and accordingly only the high peak intensities are provided for the 2P activation process. 
         [0186]    As shown in (11), in a telescopic radiation path a reconstruction of the short pulse only occurs in the sample (the image level of the telescope). 
         [0187]    An SLM as the modulator for the split wavelengths is positioned in an interim image of the display, with here the original pulse form being reconstructed. 
         [0188]    ROI is created by a selective switching of the SLM elements. 
         [0189]    Here, the ROI functionality can now also occur in the wide-field excitation (in addition to the switching). For this purpose, the laser wide-field excitation must also be guided through the SLM or be given a separate SLM; then even the separate definition of ROIs for excitation and switching is possible. This embodiment is shown in  FIG. 8  for the case of a 2-color excitation and detection. 
         [0190]    Alternatively to the SLM, a DMD (digital micro-mirror device) array may also be used. The design is similar to  FIG. 8 , however now the DMD array must be used in a reflection (again in an interim image). 
         [0191]    Astigmatism/cylinder lens detection diagram with 2P switching by point scanner 
         [0192]    The design is equivalent to  FIGS. 6 ,  8 ,  9 , however with cylinder lens(es) ZL in the detection radiation path instead of Bt 1  and/or Bt 2 . 
         [0193]    This is optionally marked in  FIGS. 6 ,  8 ,  9  by an arrow. 
         [0000]    Bi-planar Detection Diagram with 2P Switching Via Rotating Micro-lens Disk 
         [0194]      FIG. 9  shows a depth-selective 3D high-resolution microscope (schematically) with a scanning micro-lens disk (SML). The references are equivalent to those in  FIG. 8 . 
         [0195]    The irradiation occurs by 2P laser L 1 , for example at approx. 800 nm (e.g., ps laser diode). After the beam adjustment to a micro-lens array (scanning micro-lens array) (SML) the foci of the (rotating) micro-lenses are displayed in the sample via an interim image with a SLM located therein. This way, a rapidly scanned  2 P point source is realized for switching the fluorophores with ROI-functionality via the SLM. 
         [0196]    Additionally, a decoupling of the laser wide-field irradiation occurs to excite the fluorophores via dichroit  1  (D 1 ). Detection and z-information are according to the above examples. 
         [0197]    Advantages are:
       the speed   and the confocal depth discrimination at wide-field radiation, combined with   ROI functionality by SLM or DMD.
 
Confocal High-resolution Microscope with Line Scanner
       
 
         [0201]    In principle, the localization-based high-resolution methods described cannot be performed with a standard LSM: the sub-PSF-precise localization must occur by the simultaneous spatial oversampling by the camera pixels, because the molecules are fluorescent only for a limited period of time and/or activated (stochastically) at unpredictable points of time. Therefore any method is excluded in which the focal point of the molecule is determined sequentially (grid method). 
         [0202]    In the wide-field imaging as practiced in prior art again the desired depth selection is missing by a confocal illustration of a LSM. 
         [0203]    The arrangement presented here comprising a particular line sensor ( FIG. 10 ) and a respective microscope ( FIG. 11 ) allows, however, the advantageous combination of confocal line scanning and localization-based high-resolution and this way allows a PALM-similar high-resolution microscopy method in connection with the depth selection of a confocal microscope. 
         [0204]    In order to increase the penetration depth the line radiation for switching and/or excitation can also occur by a multiphoton process. 
         [0205]    This approach differs from the one of  FIG. 6  such that here depth selection is achieved by a confocal line display. 
         [0206]      FIG. 10 :  a ) shows a principle of the localization-based confocal high-resolution microscopy with a line sensor,  10   b ) a sensor with an engaged pixel structure,  10   c ) an example to realize an engaged pixel structure with existing pixel geometries by way of masking. 
         [0207]      FIG. 10   a ) explains the principle based on a 2-line sensor (for example the sensor of a cell camera as frequently used in machine vision). 
         [0208]    The lateral localization (along the cell direction) can occur as known from focal imaging or fitting a suitable function (1D Gauss), in which the engaging pixels of both cells are to be considered, here. The localization orthogonally in reference to the line direction can occur by forming the difference signal of the respective pixels of the opposite row of pixels. The principle is here equivalent to a position-sensitive detector (PSD). In order for the PSD-function to be ensured the confocal slit diaphragm must be opened slightly wider than one airy unit. 
         [0209]    The different precisions of localization in the x- and y-direction which might develop from this approach can be adjusted by an alternative sensor design.  FIG. 10   b ) shows a potential sensor structure with two engaging combs. 
         [0210]    Here, for the determination of the y-coordinate, first the determined x-position must be evaluated and considered, because by the engaged pixel structure the PSD-function (y-focal determination) depends on the x-position of the molecule to be localized. 
         [0211]    The image of the line shall be selected such that the PSF-width is precisely equivalent to the width of a pixel line; however it is centered to the middle of the engaging comb-like structure. 
         [0212]      FIG. 10   c ) shows an alternative suggestion for realization based on quadratic pixel geometries of prior art. Here, for example a normal sensor can be rotated by 45 degrees in order to yield this pixel orientation. In principle, an area sensor 
         [0213]    may also be used for this purpose, which is masked except for the desired line of detection. This mask can also be embodied as a variable diaphragm. 
         [0214]    According to the invention several, advantageously two, rows of pixels are used for assessment. In  10   a )- c ) one molecule is schematically drawn in dot-dash lines, which is detected by several detector elements. 
         [0215]    For this purpose, the existing slit diaphragm in a line scanner is opened slightly in order to simultaneously detect two rows. In  10   a  and  b  the slit image is shown, detected here. 
         [0216]    As an example,  FIG. 10   c ) shows the detected area slightly lighter than the area masked, located above and below this. 
         [0217]    By the mutual subtraction of advantageously four detector elements, which are located side-by-side of each other and each detecting a signal, the focal point of the detected molecule can be precisely determined (by the amount and algebraic sign of the result of the subtraction). 
         [0218]    Added here is depth information comprising form and size, for example using the above-cited method. 
         [0219]      FIG. 11  shows schematically a potential embodiment of a depth-selective high-resolution microscope based on the sensor principle. 
         [0220]      FIG. 11  shows a depth-selective 2D high-resolution confocal microscope (schematically) with a line scanner, for example for two-color detection. Switching and excitation lasers (L 1 -L 3 ) are brought into a linear form via a beam former (SF) and scanned in a spatial direction (scanner Sx). In the detection the line is confocally displayed by appropriate slit diaphragms on the line detectors (Ld 1 , Ld 2 ), as described in the above text. D 1 -D 4  are dichroitic beam splitters and/or combiners. 
         [0221]    The line for irradiation is generated by the beam-forming optic SF (cylinder lens) and scanned in one spatial direction (scanner Sx). The fluorescence is confocally displayed via slit diaphragms B 1  and B 2  (here an exemplary embodiment with two color channels) to the line detectors Ld 1  and Ld 2  shown sketched in  FIG. 10 . 
         [0222]    In  FIGS. 12-14  advantageous variable image modules Bt 1 , Bt 2  (see, e.g.,  FIG. 6 ) are sketched for z-splitting. 
         [0223]    The reference characters represent in detail:
       E 1 , E 2 : object levels   BM: image splitter module   O: lens   Dic 1 : primary color splitter   L 1 : light source   EF: emission filter   TL: tubular lens   SP: deflection mirror   B: diaphragm (telecentric diaphragm)   L 1 , L 2 : group of lenses   Dic  2 : color splitter for optional masking   BS: beam splitting cube   P 1  dual deflection prism, adjustable perpendicular in reference to the optic axis   P 2  deflection prism   DE wide-field detector   S 1 , S 2 : sensor halves   e 1 , e 2  image levels       
 
         [0241]      FIG. 12  shows a wide-field radiation path with a widened light source and a spatially resolving area detector, for example a CCD camera. 
         [0242]    The light of the light source L 1  reaches (reflected) the sample Pr via Dic  1  and the lens O. The reflected and fluorescent sample light travels via the lens towards detection. 
         [0243]    At the splitter Dic  1  and via the filter EF a selection occurs of the desired light, here a suppression of the reflected light; i.e. only the fluorescent light travels in the direction of detection. 
         [0244]    Via SP, L 1 , L 2  the sample light reaches the arrangement according to the invention comprising BS, P 1 , and P 2  and then the detector DE. 
         [0245]    E 1  and E 2  are different object levels in the sample Pr. 
         [0246]      FIG. 12   a  shows an enlarged detail of the variable image splitter module BM according to the invention. An image of the sample Pr is split via the beam splitter cube BS into two partial images on the detector DE. The prism P 1  is displaced in a motorized fashion perpendicular in reference to an optic axis in order to adjust the splitting of the two object levels. Via P 2  (fixed) the displaced partial image in turn is reflected into the penetrating radiation path, spatially displaced in reference to the penetrating radiation path, i.e., laterally off-set in reference to DE. 
         [0247]    Based on the image levels e 1 -e 3  and the very same object level in Pr, for example E 1 , the invention is explained in greater detail: 
         [0248]    The dot-dash position of P 1  indicates the shortest possible position of BM; the image level e 3  yielded here would be positioned behind the detector DE. 
         [0249]    The image level e 2  of the partial image, not deflected, is located on the sensor (partial image S 2  of the sensor). By displacing the prism P 1  towards the outside into the lower position in  FIG. 12   a  the path is extended and the image level e 1  moves (opposite the radiation path) towards the front. Here, it follows that an object (molecule) located in the object level 
         [0250]    E 2  is displayed focused on the sensor half S 2  and blurred on the sensor half S 1 . This applies equivalently for an object located in the object level E 1  and/or between E 1  and E 2 . 
         [0251]    E 1  would be focused on S 1  and E 2  would be focused on S 2 ; everything else would be blurred using the same prism position.) 
         [0252]    In  FIG. 12   b , in an enlarged fashion, point images of molecules  1 ,  2 ,  3  of the object levels E 1 , E 2  are each shown on the sensor halves S 1  and S 2 . 
         [0253]    It is discernible that in E 1  and E 2  molecules ( 1  in E 1  and  3  in E 2 ), each arranged in different levels, are detected focused in S 1  (molecule  1 ) and S 2  (molecule  3 ), while molecule  2  respectively is detected blurry because it is obviously located between E 1  and E 2 . 
         [0254]    Therefore, from the size of the molecules on the detector sections deductions can be drawn concerning their precise location in the z-direction in the sample. 
         [0255]    Additional radiation paths of two molecules are indicated in the drawing, with both of them being located in the object level E 2 . 
         [0256]    The diaphragm B defines the detail reduced by half (equivalent to the size of the sensor halves S 1  and S 2 ) and prevents light from outside this area from impinging the two partial radiation paths. 
         [0257]    The splitting into two partial images occurs at the 50/50 radiation splitter cube BS. 
         [0258]    The objective to focus the undeflected partial image into an image level different from the directly displayed partial image is fulfilled by the prism P 1 . This prism P 1  extends the focus of the respective bundle compared to the respective path through the air; therefore it shall preferably be embodied from highly diffractive glass, in order to obtain an operating range as large as possible. These bundles are then once more directly reflected via the prism P 2  parallel to the direct radiation path and deflected to the image sensor DE in section S 1 . 
         [0259]    With the focal extension after the last optic/lens L 2  in the prism P 1 , by selecting the length of the prism, it can be ensured that, in spite of a mandatorily longer radiation path of the deflected partial image, both partial images of different object levels E 1 , E 2  are simultaneously displayed focused on the detector DE. 
         [0260]    By displacing the prism P 1  perpendicular in reference to the optic axis of the direct radiation path the z-displacement of the focal areas of the two partial images of different object levels can be adjusted in the sample. 
         [0261]    This way, the splitting can be advantageously adjusted, for example, to different lenses. 
         [0262]    This is possible without any secondary focusing of the system, because the system has been designed such that an adjustment of “zero” can be set and particularly when observing the surface of the cover glass the second focal area can be moved into the sample (and not into the cover glass). 
         [0263]    In another arrangement, in which the glass path of the prism P 1  is waived and only two deflection mirrors are used, jointly displaceable perpendicular in reference to the optic axis, two different object levels can also be displayed focused advantageously on two different object levels on the camera. 
         [0264]    However, due to the lateral path the zero compensation would not be achieved and with the lateral displacement of the mirrors it would further move into the “cover glass.” In order to vary the second object level in the sample space then the splitting of the object level would have to be adjusted via the mirrors and then refocused at the lens such that the cover glass is once more focused via the deflection on the detector. However, this arrangement described is still within the scope of the invention disclosed here. 
         [0265]    An exchange of the prism P 1  for another prism with different glass paths is possible, according to the invention. 
         [0266]    The illustration, from the object to the detector through the lens L 2  in connection with the optic L 1  left in reference to the beam splitter Dic  2  is overall advantageously telecentric. 
         [0267]    An adjustable, preferably rectangular diaphragm B in an interim image serves to define a rectangular image area, split via P 1 , P 2 , for suppressing fluorescent and diffused light from the field ranges outside this new field. 
         [0268]    For multi-color experiments a second emission wavelength can be deflected via the color splitter Dic  2  and by the use of a second, preferably identical z-splitting module and another detector this radiation path can also be used for high-resolution localization. 
         [0269]    Potential errors of color length of the lens or other chromatic errors influencing the z-localization of the individual molecules can be compensated by adjusting the splitting for the second color channel. 
         [0270]      FIG. 13   a ) shows another advantageous embodiment of a module for splitting a camera image into 4 partial images of identical intensity for z-high resolution. 
         [0271]    In  FIG. 13   a  left, an embodiment is shown with separate mirrors and beam splitters. A first splitter T 1 , here 25/75 splitting regarding transmission and reflection, guides a portion of the light to the area Q 1  of the detector DE 4 . 
         [0272]    A second portion is T 1  reflected and guided via the mirror S 1  in the direction of a second splitter T 2  at a ratio of 66/33. It allows a portion of the light to pass in the direction of the area Q 2  of DE  4  and reflects a portion in the direction of a mirror S 3 , which deflects the light in the direction of the 50/50 splitter T 3 . A portion passes through T 3  to the area Q 3  of DE  4  and a portion is deflected via S 3  in the direction of the area Q 4  of DE  4 . 
         [0273]    By the splitting of the z-levels in the monolithic design by one “cube length” each of a beam splitter cube four evenly displaced image and/or object levels develop, with the quadrant Q 1  of DE  4  seeing the image in the direct passage, quadrant Q 2  an image displaced by a “standard distance” (=one beam splitter cube length in the monolithic design, right), quadrant Q 3  by two, and quadrant Q 4  by 3 distances. 
         [0274]    The splitter ratios of the 3 beam splitters shown here, without any limitation, ensure that all 4 quadrants preferably detect the same intensity. 
         [0275]    In  FIG. 13   b  a monolithic embodiment with 3 beam splitter cubes is shown for T 1 -T 3  and  3  mirrored prisms for S 1 -S 3  from the side, from the top, and in a perspective. 
         [0276]    The arrangement in  FIG. 3   a ) and b) is advantageously also suitable for a telecentric detection beam path as described according to  FIG. 12 . 
         [0277]    The advantages of the arrangement according to the invention in  FIG. 13  provides a more precise z-determination in a larger operating range. This is accomplished by four images of four sample levels so that four support points are provided for the z-determination. 
         [0278]    The square sensor format of the high-sensitive EMCCD cameras is further used more efficiently. 
         [0279]    In  FIG. 14  an advantageous combination occurs of an embodiment according to  FIG. 13  with displaceable prisms according to  FIG. 12 . 
         [0280]    The right side shows a view from the direction of the sensor, the left one a top view. 
         [0281]    Instead of the mirrors S 1 -S 3 , in  FIG. 14  here prisms  1 - 3  are provided, which define a radiation path parallel in reference to the optic axis through a medium of higher optic density. Compared to  FIG. 13  here additional deflection elements  1 - 3  serve to redeflect [the beams] into the respective partial radiation path. 
         [0282]    Each of the three prisms  1 - 3 , adjustable perpendicular in reference to the optic axis (direction of radiation), is provided for one quadrant Q 2 - 4  of the sensor ( FIG. 13 ); the fourth quadrant Q 1  is irradiated by the direct passage. Each prism could now be adjusted to a different displacement of levels. 
         [0283]    While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

Technology Classification (CPC): 6