Abstract:
The invention relates to a device ( 10; 210; 310; 410; 510 ) for tomographic image recording. The device comprises a sample retainer ( 14 ), a light source ( 12 ), and a detector unit ( 16 ). The light source ( 12 ) is designed to produce a pencil beam ( 38 ), which has a beam direction ( 40 ) and which passes through a sample volume of the sample retainer ( 14 ) provided in order to accommodate a sample, and has an optical control element ( 30 ), which is able to move the pencil beam ( 38 ) passing through the sample retainer ( 14 ) transversely to the beam direction ( 40 ) while the beam direction ( 40 ) remains substantially unchanged. The detector unit ( 16 ) is designed to detect at least a portion of scattered radiation ( 64; 64 ′) escaping from a section of the pencil beam ( 38 ) within the sample volume or the sample retainer ( 14 ) in a non-spatially-resolved manner.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a National Stage application of PCT International Application Serial No. PCT/EP2011/073077, filed Dec. 16, 2011, which claims priority from German Application No. 10 2010 063 412.3, filed Dec. 17, 2010. 
     TECHNICAL FIELD 
     The invention relates generally to a technique for acquiring a sectional image or a three-dimensional image. In particular, the invention relates to an apparatus for tomographic acquisition of an image of a spatial object. 
     TECHNICAL BACKGROUND 
     Resolution and contrast of existing techniques for spatial image acquisition, particularly for microscopy of living organisms, have been significantly improved in recent years. Widespread techniques with high resolution, such as the confocal microscopy described in printed publication DE 43 26 473, are based on the spatially resolved acquisition of an overlap of illumination volume and acquisition volume. The volume of the overlap determines the resolution. Alternative processes, such as optical coherence microscopy (OCM) and optical coherence tomography (OCT), determine the axial depth interferometrically by means of light of short coherence length. More recent techniques, such as the optical projection tomography (OPT) described in Science 296, 541 (2002) by J. Sharpe et al., transirradiate the specimen volume at a plurality of different angles and attain a dependent resolution from 1 μm to 5 μm. 
     Document WO 2004/020997 A1 describes an apparatus with a rotatable object stage for rotating the specimen to be imaged. A main detector acquires only light that leaves the specimen parallel to the light-incidence direction. In further embodiments, a main detector and a plurality of auxiliary detectors have been arranged in a one-dimensional or two-dimensional matrix in the beam direction downstream of the specimen with a view to angle-resolved acquisition of light, whereby light deflected from the specimen is acquired by the auxiliary detectors. 
     Document US 2007/0109633 A1 describes a microscope that transilluminates a plane in the specimen volume with a set of parallel beams of light. The illumination plane that is formed in this manner is acquired in its two dimensions by a CCD camera at a right angle to the illumination plane in spatially resolved manner. In a further embodiment, two illumination planes are irradiated. By superimposition of two coherent illumination planes, the intensity distribution in the superimposed illumination plane is structured by interference patterns with a view to improving the positional resolution in the illumination plane. With a view to spatial image acquisition, the illumination plane and the specimen are moved towards one another. A further embodiment illuminates the specimen substantially one-dimensionally and acquires the specimen in this dimension in spatially resolved manner with a linearly increased number of pixels. 
     Document US 2007/0274580 A1 describes a technique for optical tomography. Fluorescent proteins in a specimen are excited by incident light. The exciting light in the specimen and the fluorescent light are diffuse. A spatially resolved image is acquired and processed. 
     The science publication “Forward and Inverse Calculations for 3-D Frequency-Domain Diffuse Optical Tomography”, B. W. Pogue et al., SPIE Vol. 2389, pages 328-339, describes a process of diffusive optical tomography (DOT). For a simulation of the process it is assumed that a plurality of detectors have been arranged around an object on a circle. 
     The known techniques for spatial image acquisition can, by reason of a radiation intensity acting on the specimen, distort the image acquisition, change the specimen in uncontrolled manner or, particularly in the case of in vivo microscopy, destroy the specimen. Also, the technical effort in order with the prior techniques to diminish the radiation intensity with comparable image results may rise considerably. 
     SUMMARY 
     The object underlying the invention is to make available an improved apparatus for tomographic image acquisition. 
     The object is achieved by an apparatus for tomographic image acquisition having the features of Claim  1 . 
     An apparatus for tomographic image acquisition may include:
         a specimen holder,   a light-source which has been designed for generating a pencil beam with a beam direction traversing the specimen holder, and which exhibits an optical control element which is able to offset the pencil beam traversing the specimen holder at right angles to the beam direction, with substantially unchanged beam direction, and   a detector unit which has been designed for non-spatially-resolved acquisition of at least a portion of a scattered radiation escaping from a section of the pencil beam within the specimen holder.       

     The specimen holder may provide a specimen volume for receiving a specimen. The pencil beam may traverse the specimen holder, the specimen volume and/or the specimen. 
     The apparatus can acquire the scattered radiation escaping by reason of an individual pencil beam (at right angles to the beam direction). In comparison with conventional techniques, in this way a considerably larger proportion of the scattered radiation is capable of being acquired by the detector unit. Accordingly, the signal-to-noise ratio of the acquisition can be significantly improved. Also, a radiation intensity (with identical or improved image quality) introduced by the pencil beam can be diminished. In addition, a signal mixing of pencil beams that have been offset (parallel) relative to one another (i.e. various measuring positions) can be excluded. Also, the specimen volume can be acquired with a uniform sensitivity, insofar as the same detector unit acquires all the measuring positions. In particular, ring artefacts (such as frequently arise in the course of the tomographic reconstruction of a spatially resolved acquisition) can be avoided. In comparison with the spatially resolved acquisition of conventional techniques, the acquisition by the detector unit may be regarded as “zero-dimensional”. Furthermore, a more homogenous (in effect) illumination of the specimen volume, in particular at right angles to the beam direction, in comparison with a simultaneous illumination (which, for instance, employs a diffuser) can be obtained. Advantageously, for an excitation of the scattered radiation that is as homogeneous as possible the light-source comprises a laser that is stable (over an image acquisition time) by way of radiation-source. 
     Generally, the radiation-source may be substantially spatially coherent, in particular punctiform. A preferred radiation-source is a laser. For example, the radiation-source may be a source of fluorescent light, preferentially a superluminescent diode (SLD) with a small exit aperture. A diameter of the exit aperture may have been defined to be so small that the exit aperture is at least substantially diffraction-limited. Preferentially, the diameter of the exit aperture corresponds (at most) to twice the diffraction limitation. 
     “Scattered radiation” encompasses any radiation brought about by (electromagnetic, quantum-physical or thermal) interaction of the pencil beam with a specimen received in the specimen holder (in particular with the pigments, scattering centres, molecules or atoms thereof). In particular, the term “scattered radiation” encompasses radiation brought about by fluorescence, Raman scattering, Rayleigh scattering and Mie scattering. Furthermore, the scattered radiation may have been defined by a scattering direction deviating from the beam direction of the pencil beam, by a scattered-radiation characteristic (according to a differential scattering cross-section) or by a wavelength different from the pencil beam. 
     “Scattered radiation escaping” from the pencil beam may (exclusively) encompass scattered radiation scattered out of the beam direction. In the case of fluorescent radiation, scattered radiation in the beam direction can also be acquired by means of a dichroic filter. 
     In the case of fluorescent radiation by way of scattered radiation, “non-resonant fluorescence” encompasses the case of a fluorescence wavelength (of the fluorescent radiation emitted as scattered radiation) that is longer than the excitation wavelength (of the pencil beam radiated in for the purpose of excitation). 
     The non-spatially-resolved acquisition may be non-spatially-resolved in the beam direction. The non-spatially-resolved acquisition may furthermore be non-directionally resolved. Preferentially, the non-directionally-resolved acquisition is non-directionally resolved with respect to a specimen exit direction. 
     The specimen holder may be transparent to the beam at least in one section (also designated as a “window”) of the specimen holder. 
     The image acquisition can be repeated (by rotation about the axis of rotation) in various rotary positions (i.e. various directions of incidence) of the pencil beam (relative to the specimen holder). Given lesser complexity (of a specimen object to be represented), a number of the various different rotary positions may be smaller. In this way, in the case of a two-dimensional (at most) specimen object two different rotary positions can be acquired (and a specimen image can be reconstructed by triangulation). The axis of rotation may pass through the specimen holder. This includes the case where only an imaginary rectilinear extension of a structural axis of rotation passes through the specimen holder. 
     The light-source may be stationary, and the specimen holder may be rotatable about the axis of rotation. Alternatively, the specimen holder may be stationary and the light-source may be rotatable about the axis of rotation. 
     The pencil beam may exhibit a defined beam width within the specimen holder. The pencil beam may be substantially free from divergence within the specimen holder. The pencil beam may exhibit a beam waist within the specimen holder having a Rayleigh length that is substantially equal to or greater than (half) the length of the acquired section. 
     The length of the acquired section may be greater than the beam width by a multiple. The beam width may be smaller by a multiple than one or all of the internal dimensions of the specimen holder at right angles to the beam direction. The acquired section may comprise the entire pencil beam traversing the specimen holder. 
     The light-source may furthermore have been designed to generate the pencil beam in the visible spectrum, in the ultraviolet spectrum, or in the infrared spectrum. Generally, the pencil beam may lie within the electromagnetic spectrum having (vacuum) wavelengths from 300 nm to 2000 nm. This spectrum is suitable for non-destructive acquisition of an image of a specimen of deoxyribonucleic acid (DNA). A preferred spectral range of the pencil beam encompasses 323 nm to 750 nm or 355 nm to 850 nm (in the case of fluorescent radiation by way of scattered radiation). In conjunction with absorption tomography (by means of a transmission photo-detector), the pencil beam may (additionally) exhibit radiation from 200 nm to 323 nm (or from 200 nm to 355 nm). Furthermore, the pencil beam may excite the scattered radiation at least partly in the extreme-ultraviolet spectrum (XUV) or in the far-infrared spectrum (FIR). In the case of a water-free specimen, the pencil beam may (additionally) exhibit radiation within the range above 2000 nm. With a view to multi-photon excitation of the fluorescent radiation (in short: “multi-photon fluorescence”), each wavelength specification above (of a spectral range boundary) can be multiplied by a factor of 2 (in the case of a two-photon excitation), by a factor of 3 (in the case of a three-photon excitation), etc. With a view to multi-photon excitation, the light-source may emit brief intense pulses. A femtosecond laser or a (Q-switched) laser with pulse durations of a few picoseconds can be employed as radiation-source for multi-photon excitation. The multi-photon fluorescence can enable the acquisition of a greater depth (in the beam direction). 
     Alternatively or in supplement, the light-source may have been designed to generate, by means of the pencil beam, second harmonics (i.e. frequency doubling), or high harmonics, which are designated here comprehensively as “higher harmonics”, in the specimen holder (i.e. in the specimen employed in the given case). This process (mostly designated in the technical terminology as higher-harmonic generation or HHG) can coherently transform several (two, three or more) photons (of the pencil beam) into one photon. The “scattered radiation” resulting therefrom may exhibit a higher frequency, (by approximation) higher by an integral multiple, (and correspondingly shorter wavelength) than the pencil beam. The direction of the “scattered radiation” of the HHG may, in principle, be in the beam direction (of the pencil beam) or opposite to the beam direction. The “scattered radiation” of the HHG is capable of being generated along the section of the pencil beam within the specimen holder (and not only at a focal point). HHG may also occur without addition of so-called “markers” (to the specimen). Preferentially, for the HHG the light-source for generating the pencil beam has been designed with circular polarisation. The “scattered radiation” of the HHG may furthermore generate (secondary) scattered radiation at scattering centres within the specimen holder (i.e. in the specimen employed in the given case). Advantageously, by virtue of the circularly polarised pencil beam the (secondary) scattered radiation is capable of being generated at scattering centres independently of a spatial orientation of the scattering centres. 
     The detector unit includes at least one scattered-radiation collector which has been designed, or which have together been designed, to acquire the scattered radiation within a solid angle about the specimen holder of at least 0.12 π sr (or at least π/8 sr), as an example about 0.4 π sr, and preferentially at least 2π sr. The acquired solid angle of 0.12 π sr may correspond to a (maximal) photon efficiency of 3%. Furthermore, the acquired solid angle of 0.12 π sr may correspond to a numerical aperture of about NA=0.5 (using an immersion liquid). In air, a numerical aperture of NA=0.5 may correspond to an acquired solid angle of 0.28 π sr (or a maximal photon efficiency of 7%). The scattered radiation acquired in this way within the solid angle may be acquired coherently or incoherently. Advantageously it is (possible but) not necessary to acquire the scattered radiation coherently. In particular, radiation paths of the acquired scattered radiation may also exhibit differing optical path lengths, which differ by more than one coherence length. 
     The detector unit may furthermore include as a signal generator a semiconductor detector and/or a photomultiplier tube (PMT). The semiconductor detector may be a phototransistor or a photodiode, preferentially an avalanche photodiode (APD). 
     The specimen holder may have been designed to receive a liquid (viscous) specimen environment. The specimen holder and/or a liquid (viscous) specimen environment within the specimen holder may exhibit a refractive index adapted to the specimen. 
     A first collimator of the scattered-radiation collector may include a strongly refracting lens or a condenser lens which has been designed to collimate the acquired scattered radiation. Alternatively or additionally, the scattered-radiation collector may exhibit as first collimator a tapered light guide (tapered light pipe, TLP) which has been designed to collimate the acquired scattered radiation. The light guide may be a reflection-coated (on the inside) optical hollow-core conductor. Alternatively, the light guide may be a solid glass rod, so that the scattered radiation is guided by total reflection (for instance, to the air surrounding the solid glass rod). Alternatively or in supplement, the solid glass rod may have been vapour-coated with a dichroic layer. The dichroic layer can reflect the scattered radiation for steep angles of incidence (i.e. small angles of incidence relative to the normal to the inner surface of the optical hollow-core conductor). This can enable reflection (on the dichroic layer) to occur for steep angles, and total reflection to occur for shallow angles (i.e. for large angles of incidence with the normal to the inner surface of the optical hollow-core conductor). 
     The detector unit may furthermore exhibit one or more exchangeable fluorescence filters which are substantially transparent to fluorescent radiation (by way of scattered radiation). The fluorescence filter(s) may be substantially opaque within the bandwidth of the pencil beam. The fluorescence filter may be a colour filter or a dichroic filter. 
     The filter-option unit may include the several exchangeable fluorescence filters. The filter-option unit may have been designed to arrange the fluorescence filters optionally (individually) between the first collimator and the signal generator. In this way, the filter-option unit may include a rotatable wheel disc in which the several fluorescence filters have been set circumferentially. The exchangeable fluorescence filter may be capable of being chosen by the rotation. Alternatively, the filter-option unit may be a displaceable slide, along which the several fluorescence filters have been set. The exchangeable fluorescence filter may be capable of being chosen by the displacement. 
     The detector unit may exhibit a second collimator which has been arranged between the fluorescence filter and the signal generator for the purpose of focusing the acquired scattered radiation. 
     The detector unit may include several scattered-radiation collectors. The detector unit may furthermore include a signal generator assigned to all the scattered-radiation collectors, or several signal generators each assigned to one of the scattered-radiation collectors. Signal outputs of the several signal generators can be added up. The signal outputs can be added up in analogue manner, preferentially by the signal outputs having been interconnected. Alternatively or in supplement, the signal outputs can be added up digitally, preferentially by an analogue-to-digital converter having been connected to or being capable of being connected to each of the signal outputs for the purpose of separate digitisation, and by a computing unit having been connected to or being capable of being connected to the analogue-to-digital converter for the purpose of addition of the digitised signal outputs. 
     The scattered-radiation collector may furthermore exhibit an integrator at least substantially surrounding the specimen holder. An inner surface (facing towards the specimen holder) of the integrator may be a diffusely reflecting inner surface. The inner surface of the integrator may exhibit a cuboid shape. Alternatively, the integrator may be an Ulbricht sphere. A linear dimension of the integrator, preferentially an edge length or a diameter of the integrator, may be so small that a light propagation time corresponding to the linear dimension is shorter than a half-life of the fluorescent radiation excited by the pencil beam. In this connection, the linear dimension may furthermore have been multiplied by an (average) number of reflection events or scattering events (within the integrator). The half-life of the fluorescent radiation (i.e. a fluorescence lifetime) may in this way be capable of being acquired, particularly if the temporal broadening (of a signal) of the scattered radiation by virtue of variably long light propagation distances is small in comparison to the half-life. The first collimator may for this purpose have been arranged to receive scattered radiation at a measuring aperture in the integrator arranged substantially perpendicular to the beam direction. 
     The optical control element of the light-source may include:
         at least one rasterisation mirror, and   one or more actuators coupled with the at least one rasterisation mirror, for moving the rasterisation mirror.       

     In particular, the optical control element may have been designed as a relay lens system with:
         a first rasterisation mirror actuated by a first actuator for offsetting the pencil beam in a first rasterisation direction,   a second rasterisation mirror actuated by a second actuator for offsetting the pencil beam in a second rasterisation direction substantially perpendicular to the first rasterisation direction, and   a lens, two lenses or a lens system (between the first rasterisation mirror and the second rasterisation mirror) which has/have been designed to image the first rasterisation mirror onto the second rasterisation mirror.       

     The optical control element may be voltage-controlled, preferentially capable of being actuated piezoelectrically. In this way, a piezoelectric crystal may have been employed as actuator. Alternatively, the optical control element may be current-controlled, preferentially by means of a moving-magnet galvanometric drive or a moving-coil galvanometric drive. In this way, a galvanometric drive (also designated as galvo-scanner) may have been employed as actuator. Alternatively, the optical control element (for each of the two rasterisation directions) may exhibit a polygon scanner. The polygon scanner includes a mirror polygon and a motor, on the axis of which the mirror polygon has been rotatably arranged. The mirror polygon is a cylinder with a regular polygon as base, the lateral faces of which are silvered. Furthermore, the optical control element (for each of the two rasterisation directions or for a fast-rasterisation direction) may exhibit an acousto-optic modulator (AOM). As optical control element, the AOM is also designated as acousto-optic deflector (AOD). The AOM includes a beam-transparent solid body in which a piezoelectric element arranged at right angles to the beam direction is able to generate an ongoing soundwave having an ultrasonic frequency. With a view to fast rasterisation (i.e. for offsetting the pencil beam), the ultrasonic frequency is variable. 
     The one or more actuators may have been designed to move the rasterisation mirror substantially perpendicular to the beam direction, or to swivel it about a swivel axis substantially perpendicular to the beam direction. For the purpose of offsetting the pencil beam in a first rasterisation direction and in a second rasterisation direction that is not parallel to the first rasterisation direction, a first actuator and a second actuator of the several actuators may have been provided. The first rasterisation direction is preferentially (substantially) parallel to the axis of rotation. 
     The light-source may furthermore exhibit a radiation-source and a beam-shaping lens system which has been arranged between the radiation-source and the optical control element. The beam-shaping lens system may include a telescopic lens system for beam enlargement. Furthermore, the beam-shaping lens system may include an annular diaphragm and an axicon for generating a Bessel beam by way of pencil beam. Alternatively or in supplement, the beam-shaping lens system may include a spiral phase plate (vortex phase plate) for generating the pencil beam with optical vortex or Laguerre-Gauss mode (doughnut mode). 
     The light-source may furthermore exhibit a focusing lens system which has been arranged between the optical control element and the specimen holder. The focusing lens system may comprise a focusing lens or a focusing-lens system for forming the pencil beam (with the defined beam within the specimen holder). Preferentially, the focusing lens system exhibits a telescopic lens system and a focusing lens. To the advantage of a higher resolution, the focusing lens system can widen the beam (downstream of the rasterisation mirrors). In this way, a width of the rasterisation mirrors may be smaller than the beam width. The focusing lens system can radiate the pencil beam into the specimen holder with a larger numerical aperture. In this way, an incident numerical aperture of NA E =0.2 is attainable. (The incident numerical aperture, NA E , is not to be confused with the acquired numerical aperture, NA.) A first focal plane of the focusing lens system may include (substantially) a position of the rasterisation mirror. A second focal plane of the focusing lens system may include (substantially) a position within the specimen holder, preferentially a position of the beam waist or of the axis of rotation within the specimen holder. 
     The apparatus may furthermore include a computing unit which has been connected to or is capable of being connected to the signal generator of the detector unit for the purpose of transmitting a data record comprising intensity data from the signal generator to the processing unit. The intensity data may represent intensities of the acquired scattered radiation. The optical control element may have been designed to offset (controlled by the computing unit) the pencil beam (stepwise or continuously) into a plurality of different measuring positions. The data record may be a raster data record which comprises intensity data relating to each of the plurality of measuring positions. 
     The apparatus may furthermore include a motor (controlled by the computing unit) for (stepwise or continuous) rotation of the specimen holder and of the light-source relative to one another into a plurality of different rotary positions. The data record may be a tomographic data record which comprises a raster data record relating to each of the plurality of rotary positions. 
     The computing unit may furthermore have been designed to reconstruct, on the basis of the tomographic data record, a sectional image or a spatial image. The reconstructed image may represent a scattering coefficient (of a specimen in the specimen holder) and/or (in supplement) an extinction coefficient (of the specimen in the specimen holder). The reconstruction may comprise an inverse Radon transformation. Alternatively or additionally, the reconstruction may comprise an iterative reconstruction. The computing unit may furthermore have been designed to correct an extinction of the pencil beam in accordance with the (integral) Lambert-Beer law, or to take it into consideration in the reconstruction. 
     The object is furthermore achieved by a process for tomographic image acquisition having the process steps of Claim  19 . 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further advantages and features of the invention will become apparent from the exemplary embodiments described below with reference to schematic drawings. Shown are: 
         FIG. 1  a top view of a first exemplary embodiment of the apparatus for tomographic image acquisition with an integrator, 
         FIGS. 2   a  and  2   b  a top view and a side view, respectively, of a second exemplary embodiment of the apparatus for tomographic image acquisition with a reflecting specimen vessel, 
         FIG. 3  a top view of a third exemplary embodiment of the apparatus for tomographic image acquisition with a reflector, 
         FIG. 4  a top view of a fourth exemplary embodiment of the apparatus for tomographic image acquisition with several scattered-radiation collectors, 
         FIG. 5  a top view of a fifth exemplary embodiment of the apparatus for tomographic image acquisition with a collimator recess, and 
         FIG. 6  a flow chart of a process for tomographic image acquisition. 
     
    
    
     DETAILED DESCRIPTION 
     Reference symbols, relating to various exemplary embodiments, having the same two final numerals relate to similar components (interchangeable among the exemplary embodiments). 
       FIG. 1  shows an apparatus denoted generally by  10  for tomographic image acquisition. The apparatus  10  includes a light-source  12 , a specimen holder  14  and a detector unit  16 . 
     The light-source  12  includes a radiation-source  18  which emits an at least substantially spatially coherent raw beam  20 . In the exemplary embodiment that is shown, the radiation-source  18  is a laser. If a beam width w of the raw beam  20  made available by the radiation-source  18  is too small, a beam-shaping lens system  22  includes a telescopic lens system  24  which widens the raw beam  20 . 
     Irrespective of the radiation-source  18  employed, the beam-shaping lens system  22  includes a variety of diaphragms and phase masks  26  which change the raw beam  20  into a shaped beam  28 . The beam-shaping lens system  22  enables imaging properties of the apparatus  10  to be varied. In this way, a resolution attained with the apparatus  10  can be increased or can be adapted in stepless manner, in particular taking into consideration a specimen-dependent maximal beam intensity. Above and beyond this, the diaphragms or phase masks  26  project the shaped beam  28  into an optical control element  30  of the light-source  12 , arranged downstream in the beam path. 
     Optionally, a beam-splitter  32  may have been incorporated between the beam-shaping lens system  22  and the optical control element  30 , which branches off a partial beam  34  from the shaped beam  28  into a reference detector  36 . In the exemplary embodiment that is shown, a photodiode is employed as reference detector  36 . The reference detector  36  monitors, on the one hand, the stability of the laser of the radiation-source  18 . On the other hand, the reference detector  36  (as signal generator of a control circuit which is not shown) serves for selective intensity modulation of the radiation-source  18 . In connection with the intensity modulation, in principle an increased intensity for illuminating the specimen is assumed. A detector-side dynamic range (of each of the detectors described below, in particular of the detector unit  16 ) has been exhausted if the radiation-source  18  evokes a detector-side overload or signal saturation. The intensity of the radiation-source  18  is selectively reduced so far for individual partial acquisitions (ideally, for each of the measured positions, described below, of the optical control element  30 ) until no detector-side overload is present. By virtue of the intensity modulation, a generation-side dynamic range (determined by the radiation-source  18  and the reference detector  36 ) is multiplied by the detector-side dynamic range to yield a (extended and lower-noise) dynamic range of the acquisition. 
     The optical control element  30  enables the course of a pencil beam  38  through the specimen holder  14  to be offset substantially parallel to the beam direction  40 . A (transverse) measuring position of the optical control element  30  will be described in a rectangular coordinate system at right angles (or perpendicular) to the beam direction  40  by an x-offset and a y-offset. In a simplified embodiment, the optical control element  30  includes only an x-control element  42  with an x-actuator  44  which tilts an x-rasterisation mirror  46 . An x-swivel axis of the x-tilting corresponds to the y-axis of the transverse coordinate system (and is consequently perpendicular to the beam direction  40 ). In the exemplary embodiment that is shown, the optical control element  30  furthermore includes a y-control element  48  with a y-actuator  50  which tilts a y-rasterisation mirror  52  about a y-swivel axis (corresponding to the x-axis of the transverse coordinate system). In an alternative embodiment (not shown) of the optical control element  30 , a single rasterisation mirror is tilted by the x-actuator  44  and the y-actuator  50  about the x-swivel axis and the y-swivel axis, respectively. By way of actuators  44 ,  50 , use is made of galvanometric drives (also designated as “galvos” or “galvo scanners”). The actuators  44  and  50  each include an automatic control system which acquires a rotary mirror position of the rasterisation mirrors  46  and  52 , respectively, and regulates a current for attaining the desired offset. 
     The optical control element  30 , more precisely the last of the two rasterisation mirrors  46 ,  52  in the beam path, is located in a rear first focal plane of a focusing lens system  54  of the light-source  12 . The optical control element  30  deflects (by reflection) the beam path around an optical axis of the focusing lens system  54 . The focusing lens system  54  has been designed to project the pencil beam  38  into the specimen holder  14  with a beam waist in the centre of a section of the pencil beam  38  within the specimen holder (corresponding to a second focal plane of the focusing lens system  54 ). By deflection (by means of the last rasterisation mirror  52  in the beam path) in the first focal plane of the focusing lens system  54 , a telecentric projection of the pencil beam  38  (into the specimen holder  14 ) is obtained. This permits a parallel offset also for large deflection angles (corresponding to a doubled tilt angle of the rasterisation mirror  46 ,  52 ). Deviations from a parallel (to the beam direction  40 ) beam offset could make a later image reconstruction more difficult. The parallel offset of the pencil beam enables a quick (tomographic) image reconstruction by inversion of a Radon transformation. As an alternative to the tilting of the at least one rasterisation mirror  46 ,  52 , the light-source can be displaced (with respect to the specimen holder  14 ). 
     In an embodiment, not shown, of the light-source  12  without telecentric arrangement (of the optical control element  30  and of the focusing lens system  54 ), a tomographic image acquisition is likewise attainable. In this connection, the offset of the pencil beam  38  brought about by the optical control element  30  is given the beam direction  40 , at least insofar as the (transverse) offset (in the specimen holder  14 ) is small in comparison to the (optical) path length between the optical control element  30  and the specimen holder  14 . In other words, parallelism of the pencil-beam offset in the specimen holder is obtained (by approximation) for small tilt angles of the optical control element  30 . Furthermore, a (tomographic) image can also be reconstructed in the case of greater fanning of the pencil beam  38  (at the location of the specimen holder around the ideal beam direction  40  in the case of various measuring positions) by adapted direct or iterative processes. 
     A first embodiment (not shown) of the specimen holder  14  has been designed to position a specimen directly. To this end, the specimen holder  14  may exhibit a clip for the specimen, a tightening screw for clamping the specimen, or an adhesive surface for attaching the specimen. In this way, large specimens can be (slowly) rotated. A second embodiment (not shown) of the specimen holder  14  has been designed for positioning a gel volume (preferentially an agarose gel). The specimen can be enclosed in the gel volume (by casting and gelation). The third embodiment of the specimen holder  14 , which is shown, includes a glass tube filled with a refractive-index-adapted liquid, into which the specimen can be drawn. In principle, a refractive index of the specimen can be adapted as an alternative or in supplement to an adaptation (each adaptation described herein) of the refractive index of the specimen environment. For techniques for adjusting the refractive index of the specimen, reference is made to EP 1 520 173 B1 (and, in particular, paragraphs [0013] ff therein). 
     The specimen holder  14  has been rotatably arranged in a specimen vessel  56 , preferentially a cell. A liquid (which in the case of the third embodiment of the specimen vessel  14  coincides, at least with regard to its refractive index, with the aforementioned liquid) adapted to the specimen as regards its refractive index is located between the specimen holder  14  and the specimen vessel  56 . The rotation relates to a relative rotation between, on the one hand, the specimen holder  14  and, on the other hand, at least the light-source  12  and the specimen vessel  56 . In principle, a “boundary” or “interface” of the rotation extends between the specimen holder  14  and the specimen vessel  56  (particularly since the specimen vessel  56  may exhibit entrance windows and exit windows, described below, for the pencil beam  38 , the location of which, with a view to simplified reconstruction, should not be rotated in relation to the pencil beam  38 ). An axis of rotation of the rotation is vertical (perpendicular to the plane of the drawing of  FIG. 1 , corresponding to the y-direction). The (imaginary) axis of rotation of the rotation extends through the specimen holder  14 . In the exemplary embodiment shown in  FIG. 1 , the specimen holder  14  is cylindrical and the axis of rotation coincides with the cylinder axis. In a first variant, an electric drive, preferentially a stepper motor (corresponding to the stepper motor denoted by  88  in the exemplary embodiment shown in  FIG. 2 ), has been provided for rotating the specimen holder  14 . The light-source  12  and the detector unit  16  are stationary. In a second variant, the electric drive has been designed to rotate all the other components of the apparatus  10  in relation to the unmoved specimen holder  14 . 
     Particularly for a mobile apparatus  10 , all the components except the rasterisation mirrors  46 ,  52  and the specimen holder  14  have been rigidly connected to one another. Furthermore, in the second variant a stationary or immovable specimen can be examined, for instance a rooted plant, whereby the specimen vessel  56  and the entire light-source  12  rotate about the specimen. In this connection, with a view to the examination of larger specimens the specimen vessel  56  (and in the case of stationary specimens, even the specimen holder  14 ) can be dispensed with. 
     With the apparatus  10  a non-transparent specimen can also be acquired tomographically. The scattered light acquired in positionally unresolved manner can in this case be reflected substantially at the surface of the non-transparent specimen or scattered by any other effect. The non-transparent specimen may have a tomographically acquirable surface structure. For example, the surface of the specimen may exhibit an at least partially transparent coating. The scattered light may comprise scattered components and reflected components. The scattered portion may have been brought about by scattering of the pencil beam in the coating. The reflected portion may have been brought about by reflection of the pencil beam or of the forward-scattered pencil beam at the surface of the non-transparent specimen. A screw is an example of the non-transparent specimen. 
     With a view to spatial positioning of the specimen, in a further first variant or in the aforementioned first variant the specimen holder  14  (and, synchronously therewith, the specimen vessel  56 ) is capable of being transported by means of a stage micrometer in all three directions (i.e., in the x-direction, the y-direction and the beam direction  40 ). In a further second variant or in the aforementioned second variant, the specimen holder  14  (and the specimen vessel  56 ) is stationary, and the light-source  12  and the detector unit  16  (or all the other components except the specimen holder  14  and the specimen vessel  56 ) can be moved in all three directions. In a simplified exemplary embodiment (of the first variant or of the second variant), the transportability has been restricted to the vertical direction (i.e. the y-direction). 
     A tilting of the axis of rotation is present if
         (A) the axis of rotation is not perpendicular to the beam direction  40 , or   (B) the axis of rotation is not perpendicular to the x-direction of the optical control element  30 .       

     In general, the tilting of cases (A) and (B) is present in combined manner. In other words, a tilting is present if the axis of rotation is not spatially parallel to the y-axis. 
     The tilting may make the later image reconstruction more difficult or cause tilting artefacts in the reconstructed image. With a view to adjustment of the apparatus  10 , in particular with a view to compensating for the tilting, adjusting mechanisms have been provided which enable a swivelling of the axis of rotation (and hence also of the specimen holder  14 ) relative to the beam direction  40 . The aim of the adjusting mechanisms is to align the axis of rotation parallel to the y-axis as direction in space. 
     With regard to the tilting of case (A), the optical control element  30  and the focusing lens system  54  are displaceable relative to one another by means of a first adjusting mechanism. In this way, the focusing lens system  54  can be displaced perpendicular to the optical axis of the focusing lens system  54 . Alternatively, the optical control element  30  can be displaced perpendicular to the optical axis of the focusing lens system  54 . (In principle, it is also possible to displace the entire light-source, with the exception of the focusing lens system  54 .) By virtue of the displacement in the y-direction, a rising or falling course of the pencil beam with respect to the y-direction can be set. In this way, a right angle between the axis of rotation and the beam direction  40  can be set (in the plane spanned by the beam direction  40  and the y-direction). Alternatively or in supplement to the displacement, the first adjusting mechanism enables the axis of rotation to be swivelled about the x-axis. Also, a perpendicular course of the pencil beam relative to the axis of rotation can be set thereby (in the plane spanned by the beam direction  40  and the y-direction). 
     With regard to the tilting of case (B), a second adjusting mechanism enables the axis of rotation (and hence also the specimen holder  14 ) to be swivelled about the optical axis of the focusing lens system  54 . Alternatively, the optical control element  30  can also be swivelled about the optical axis of the focusing lens system  54 . As a result, the axis of rotation can be caused to coincide with the y-direction. Furthermore, the tilting of case (B) (unlike the tilting of case (A)) is also capable of being compensated by a numeric rotation of the raw data acquired (at a rotary position) in the xy-plane. 
     Inasmuch as tilting is avoided in the optical set-up, a subsequent reconstruction by layers can be decoupled. A “layer” encompasses all acquisitions at measuring positions with the same position in the y-direction (i.e. at the same “height”). The reconstruction of the tomographic image acquisition can be parallelised for various layers. To the extent that tilting is present, dependencies (between acquisitions at various positions in the y-direction) arise which do not fundamentally preclude a tomographic image reconstruction. However, consideration of the dependencies can increase the complexity of a reconstruction algorithm. 
     To the extent that two rasterisation mirrors  46 ,  52  are employed in the optical control element  30 , in a simple embodiment (of the optical control element) the optical control element  30  may have been arranged in such a way with respect to the focusing lens system  54  that the first focal plane of the focusing lens system  54  is situated between the rasterisation mirrors  46  and  52 . In this way, a fanning of the pencil beam  38  (i.e. a deviation from an ideally uniform beam direction  40 ) can be minimised. Alternatively, either the x-rasterisation mirror  46  or the y-rasterisation mirror  52  may have been arranged in the first focal plane of the focusing lens system  54 . In this way, the fanning can be restricted to the rasterisation in the y-direction or in the x-direction (and taken into consideration by the reconstruction algorithm). A further adjusting mechanism is provided, which is able to move the two rasterisation mirrors  46  and  52  (in the x-direction and in the beam direction  40 ). An extended embodiment of the optical control element  30  includes the aforementioned relay lens system. By means of the relay lens system, both the x-rasterisation mirror  46  and the y-rasterisation mirror  52  can be arranged in a respective focal plane (of the relay lens system). The last rasterisation mirror  52  in the beam path is situated, in addition, in the first focal plane of the focusing lens system  54 . In this way, a fanning in the x-direction and in the y-direction can be minimised. For simplified embodiments, individual adjusting mechanisms or all adjusting mechanisms are dispensed with. 
     The volume between the specimen holder  14  and the specimen vessel  56  has been filled up with a liquid  58  (at the height of the pencil beam  38 ) approximated to the refractive index of the specimen holder  14  (and of the specimen therein to be examined). The liquid  58  serves as immersion liquid. Inasmuch as the liquid  58  wets the specimen holder  14 , the (generally curved) surface of the specimen holder  14  as optical interface (as regards undesirable refraction or scattering) is almost eliminated. The beam which generally is weakly focused by the focusing lens system  54  is projected by a smooth glass surface  59  of the specimen vessel  56  into the specimen vessel  56 . “Smooth” means that the glass surface  59  is flat (i.e. not curved). In addition, “smooth” may encompass a slight (as far as possible) surface roughness. 
     Radiation transmitted by the specimen is projected by a second focusing lens system  60  onto a transmission photodetector  62 . A first focal plane of the second focusing lens system  60  coincides with the second focal plane of the first focusing lens system  54 . A rear second focal plane of the second focusing lens system  60  coincides with a position of the transmission photodetector  62 . In this case, an exact positioning of the focal planes of the second focusing lens system  60  is not required. The demands made of the imaging properties of the second focusing lens system  60  are less than those of the first focusing lens system  54 . 
     To the advantage of a larger dynamic range covered by the transmission photodetector  62 , and with a view to improving the dynamic range of the acquisition (without losses of speed in the course of image acquisition), the transmission photodetector  62  has a non-linear response, preferentially a logarithmic response. The “speed” of the image acquisition relates, in particular, to the exposure-time for each measuring position. Moreover, intensities that have been made logarithmic are more advantageous (since they are immediately convertible) output data for computer-aided reconstruction. 
     In conjunction with the transmission detector unit  60 ,  62 , furthermore dark-field microscopy, polarisation microscopy, interference contrast microscopy (which is also designated as differential interference contrast microscopy or DIC microscopy) or phase contrast microscopy can be realised. 
     In a simplified exemplary embodiment, the second focusing lens system  60  and the transmission photodetector  62  are dispensed with. In an additional exemplary embodiment, furthermore a diaphragm has been provided between the second focusing lens system  60  and the transmission photodetector  62  for the purpose of limiting the forward scattering. Inasmuch as (instead of the transmission photodetector  62 ) the diaphragm has been arranged in the (rear) second focal plane of the second focusing lens system  60 , the forward scattering emanating from the entire section of the pencil beam in the specimen holder  14  can be acquired separately. In the case of non-resonant fluorescence, transmission and forward scattering can furthermore be separated by a dichroic filter. 
     Scattered radiation  64  emanates from the specimen by reason of the incident pencil beam  38 . Often, the scattered radiation  64  emitted from the specimen is distributed almost isotropically (i.e. in all directions substantially uniformly). In the case of fluorescent radiation by way of scattered radiation  64 , the radiation-source  18  has been designed to generate a pencil beam  38  which excites one or more selected transitions of a fluorophore (functionally or structurally marking the specimen) in the specimen. The intensity of the re-emitted scattered radiation  64  is acquired in the form of an acquired scattering-intensity distribution, I(x,y,π), at various measuring positions in the x- and y-directions as well as various rotary positions. Generally, the acquired scattering-intensity distribution contains information (accessible through the tomographic reconstruction) about the fluorophore concentration, ρ(x,y,z), in the specimen (in the case of fluorescent radiation) or the scattering coefficient, σ(x,y,z), of the specimen (in the case of a different scattering mechanism). For a given rotary position n, the (integral) scattering-intensity distribution I(x,y,π) is an item of 2-dimensional information which results from the 3-dimensional scattering intensity σ(x,y,z) (substantially by projection in the beam direction  40  or summation along the pencil beam  38 ). 
     The scattered radiation  64  is acquired integrally by means of the detector unit  16 . The integral acquisition of the scattered radiation  64  includes the positionally unresolved and directionally unresolved acquisition of the scattered radiation  64  brought about by the pencil beam  38 . 
     The detector unit  16  includes a scattered-radiation collector  66  for integral acquisition of the scattered radiation  64  within a solid angle that is as large as possible about the specimen holder  14 . A numerical aperture NA/n (normalised to the refractive index) corresponds to the acquired solid angle. With a view to integral acquisition of the scattered radiation  64 , the specimen holder  14  (and the specimen vessel  56 ) have been arranged in the centre of an integrator  68  of the scattered-radiation collector  66 . The integrator  68  includes a capsule which is highly reflecting and strongly light-scattering in the interior. An inner surface  69  of the integrator  68  has been coated with barium sulfate (or with another scattering medium). In a simpler embodiment of the integrator  68 , the inner surface  69  exhibits a rough base, onto which a metal layer (preferentially consisting of silver) has been vapour-deposited. The exemplary embodiment shown in  FIG. 1  employs by way of integrator  68  an Ulbricht sphere. By virtue of the (multiple) scattered reflection, the scattered radiation (emanating from the specimen) is homogeneously distributed in the interior of the integrator  68 . The homogeneity has in consequence (given almost lossless scattered reflection) several technical aspects. On the one hand, an exact positioning of the specimen holder  14  (and of the specimen vessel  56 ) in the centre of the integrator  68  is not necessary in the course of design and manufacture of the apparatus  10 . In this way, hollow bodies differing from a spherical shape are suitable by way of integrator  68 . In a compact embodiment, the integrator  68  is realised by the appropriately coated specimen vessel  56 . On the other hand, the intensity of the scattered radiation at all apertures  70 ,  72 ,  74  of the integrator  68  is substantially of the same magnitude. 
     The proportion of the scattered radiation  64  that emerges at the measuring aperture  72  is connected with the entire scattered radiation  64  in accordance with an aperture ratio of the measuring aperture  72  to an entire aperture area (comprising the apertures  70 ,  72  and  74 ) of the integrator  68 . In this way, with a view to integral acquisition of the scattered radiation  64 , the entire scattered radiation  64  can be determined by acquiring the scattered radiation emerging at the measuring aperture  72 . 
     For a high signal-to-noise ratio, the measuring aperture  72  of the integrator  68  has been chosen to be as large as possible, and all the other apertures  70 ,  74  in the integrator  68  have been chosen to be as small as possible. In order to improve the aperture ratio, in an optimised variant of the exemplary embodiment merely an x-control element  42  has been provided (as described above), and the illumination aperture  70  is a slit that is as narrow as possible in the y-direction (and extending in the x-direction). Similarly, the transmission aperture  74  is a slit (as narrow as possible in the y-direction) in the x-direction. A slit height Δy amounts to 100% to 500%, preferentially about 200%, of a beam width of the pencil beam  38  at the slit. In an exemplary embodiment, the illumination aperture  70  and the transmission aperture  74  are respectively a slit with a height of Δy=0.5 mm and a width of Δx=12 mm, and the measuring aperture  72  is square with edges having a length of 10 mm. Consequently, for the exemplary embodiment an ideal efficiency corresponding to the aperture ratio amounts to 100/112=89.3%. 
     In order also to acquire measuring positions with varying y-offset in the case of the aperture ratio improved through a slit shape, a y-actuator (as an alternative to the y-control element  48  with the y-rasterisation mirror  52 ) is employed which moves the specimen holder  14  (advantageously synchronously with the specimen vessel  56 ) in the y-direction corresponding to the desired y-offset (up or down). In an alternative variant of the exemplary embodiment, the specimen holder  14  (and the specimen vessel  56 ) is stationary in the y-direction, with the light-source  12  and the detector unit  16  (or all the other components except the specimen holder  14  and the specimen vessel  56 ) being transportable up and down in the y-direction. 
     For technical reasons, an integrator  68  that is as small as possible is to be preferred to a larger integrator  68 , the advantages of which may include smaller scattering-reflection losses on the smaller inner surface  69  and narrower slits at the illumination aperture  70  and at the transmission aperture  74 . 
     The measuring aperture  72  should be equal in size to or larger than the specimen, the specimen holder  14 , or the specimen vessel  56 . (A possible specimen size may amount to about 2 mm. In the case of specimens of greater dimension, the measuring aperture  72  may also be smaller than the specimen size.) More precisely, an edge length of the square measuring aperture  72  should be greater than a length of the specimen holder  14  in the beam direction  40 . Since the homogenised scattered radiation  64  in the integrator  68  also falls onto the specimen holder  14 , in this way a portion of multiply-scattered radiation at the measuring aperture  72  (and a corresponding undesirable background signal) can be suppressed. In the case of non-resonant fluorescent radiation, the wavelength shift in addition suppresses the situation where fluorescent radiation excites further fluorescence. 
     In a further development of the exemplary embodiment, a slit width of the illumination aperture  70  and of the transmission aperture  74  is variable. Furthermore, optical internals in the integrator  68  or lens systems in the apertures  70 ,  72 ,  74  have been designed to reduce losses further at the apertures  70 ,  72 ,  74 . 
     The scattered-radiation collector  66  furthermore includes a first collimator  76  which is able to reduce the divergence of the scattered radiation emerging from the measuring aperture  72 . The first collimator  76  includes either a tapered light guide (tapered light pipe)  76   a , a strongly refracting lens  76   b , in particular a condenser lens; a concave mirror (not shown) with a central aperture; or a combination comprising two or more of these elements. By way of light guide, a reflectively coated (silvered) optical hollow-core conductor or a dielectric optical waveguide (utilising the total reflection) is capable of being employed. The first collimator  76  shown in the exemplary embodiment represented in  FIG. 1  includes the optical hollow-core conductor  76   a  and the lens  76   b . The divergence of the partly collimated scattered radiation  78 ′ at the output of the optical hollow-core conductor  76   a  is further reduced by the lens  76   b  to yield the collimated scattered radiation  78 . 
     In an alternative (not shown), the measuring aperture  72  and the light guide  76   a  have been replaced by a glass-fibre bundle. The glass-fibre bundle comprises a plurality of glass fibres which extend in stellate manner (i.e. radially) from a sphere (to this extent, replacing the integrator  68 ). The ends of the glass fibres receiving the scattered radiation  64  from the specimen holder  14  are uniformly distributed over the inner surface  69  of the sphere. The glass fibres have been designed to bring the scattered radiation to a focus in a plane  64 . Also in this alternative, in this way partly collimated scattered radiation  78 ′ is obtained which is projected by one of the aforementioned elements of the collimator  76  to yield the collimated scattered radiation  78 . 
     The collimated scattered radiation  78  exhibits a reduced divergence with a residual divergence. A sufficiently low residual divergence enables a dichroic filter to be applied to the collimated radiation  78 . Most filters are capable of being applied downstream of a first collimator  76  which attains a residual divergence of less than ±30°. When use is made of coloured-glass filters, the scattered radiation  64  is not subject to any appreciable divergence restriction. In this way, in simplified exemplary embodiments with coloured-glass filters a simple first collimator  76  (with only one of the elements) or no first collimator  76  has been provided. 
     The collimated scattered radiation  78  optionally passes through one filter of several filters which have been combined in a filter-option unit  80 , taking the form of a filter disc or filter drawer, of the detector unit  16 . In the case of fluorescent radiation, spectral regions of high transparency of the filters are each tuned to a fluorescence transition of one or various fluorophores. A resulting filtered scattered radiation  82  exhibits the intensity to be measured. 
     In additional exemplary embodiments, dichroic mirrors (not shown) are employed for the purpose of splitting various colour components into various beam paths. The dichroic mirrors are arranged in supplement to, or instead of, the filter-option unit  80 . This enables a simultaneous acquisition of the various colour components contained in the scattered radiation  64 . 
     The detector unit  16  furthermore includes a second collimator  84  and a signal generator  86  (arranged in a rear focal plane of the second collimator  84 ) for photodetection. In the exemplary embodiment shown in  FIG. 1 , the second collimator lens  84  is a converging lens, preferentially a condenser lens. With a view to optimising a quantum yield, the second collimator  84  focuses the filtered scattered radiation  82  onto the signal generator  86 , without exceeding the numerical aperture thereof. In the exemplary embodiment shown in  FIG. 1 , the signal generator  86  is a photomultiplier tube. For a beam intensity acting on the specimen that is as low as possible, the photomultiplier tube exhibits a high responsivity (and quantum yield). 
     Furthermore, the signal generator  86  is displaceable (at right angles to the direction of the filtered scattered radiation  82 ), in order to illuminate optimally an optically sensitive surface (detecting surface) of the signal generator  86 . In this way, the detector unit  16  can guide the scattered radiation  64  of the measuring aperture  72  efficiently onto the detector surface. 
     The following processes are capable of being implemented with the apparatus  10  which has been described: FLIM (fluorescence-lifetime imaging microscopy), whereby the fluorescence lifetime and the light propagation time in the integrator  68  have been matched to one another with a view to avoiding restrictions by virtue of diffuse scattering in the integrator  68 ; MPM (multi-photon microscopy); FRET (fluorescence resonance energy transfer); FRAP (fluorescence recovery after photobleaching); STED (stimulated emission depletion), whereby after the pencil beam  38  by way of exciting beam a “tube beam” by way of exclusion beam with substantially cylindrically symmetrical intensity distribution is radiated in; FLIP (fluorescence loss in photobleaching); so-called superresolution microscopy; scattered-light microscopy; possibly the following may also be capable of being implemented: STORM (stochastic optical reconstruction microscopy) and FCS (fluorescence correlation spectroscopy). 
       FIG. 2  shows a second exemplary embodiment of an apparatus  210  for tomographic image acquisition. For components of the second exemplary embodiment having the same reference symbols as in the first exemplary embodiment, what was described above applies correspondingly, particularly as regards their properties, constitution, function and their interaction. 
     The second exemplary embodiment differs from the first exemplary embodiment in that no separate integrator  68  has been provided for integrating and homogenising the acquired scattered radiation  64 . In other words, with a view to integral acquisition of the scattered radiation  64  a scattered-radiation collector  266  does not include an integrator  68 . The beam-transparent specimen vessel  256  exhibits a reflecting surface  269  on all the side surfaces (i.e. on all the surfaces except a transparent bottom surface  272 ). The reflecting surface  269  undertakes the function and action of the (diffusely reflecting) inner surface  69 . In this way, the specimen vessel  256  can also undertake the function and action of the integrator  68 . 
     A simple first embodiment of the specimen vessel  256  is an uncoated glass cell. Scattered radiation  64 , which has a tendency to be radiated to the bottom surface  272  of the uncoated cell, is conducted by total reflection at the cell/air boundary layer to the bottom surface  272 . In this way, a proportion of about 12.5% of the scattered radiation  64  is capable of being acquired. The proportion results by virtue of the angle of total reflection at the wall of the specimen vessel  256  (i.e. at the interface from cell to air). In a second embodiment of the specimen vessel  256 , a base body made of glass (on the outside or on the inside) exhibits a layer of barium sulfate or a metal layer by way of reflecting surface  269 , as described above in connection with the inner surface  69 . In a third embodiment of the specimen vessel  256 , the side surfaces of a metallic base body act as reflecting surface  269 . In a fourth embodiment of the specimen vessel  256 , the latter tallies with the specimen vessel  56  and has furthermore been set in accurately fitting manner into a mirror box (not shown) having reflecting surfaces  269 . 
     With a view to radiating the pencil beam  38 , and optionally for transmission detection, the specimen vessel  256  exhibits an illumination aperture  270  and a transmission aperture  274 , respectively. The illumination aperture  270  and, where appropriate, the transmission aperture  274  correspond (functionally) to aperture  70  and to aperture  74 , respectively. As described above in connection with apertures  70 ,  72 ,  74 , with a view to minimising radiation losses apertures  270  and  274  take the form of narrow slits in the x-direction, and the optical control element  30  rasterises the pencil beam  38  only in the x-direction. For measuring positions with y-offset, the specimen holder  14  can be moved relative to all other components in the y-direction, whereby the specimen holder  14  or the other components are stationary, as has been described above for the first exemplary embodiment. The reflecting surface  269  exhibits opposing beam-transparent slits by way of apertures  270  and  274 . In the case of the base body made of glass, apertures  270 ,  274  are uncoated. In the case of the metallic base body, apertures  270 ,  274  include plane-parallel windows. In the case of fluorescent radiation by way of scattered radiation, with a view to further reduction of radiation losses the windows exhibit a dichroic layer transmitting the exciting radiation (of the pencil beam  38 ) and reflecting the fluorescent radiation, or consist of a plane-parallel birefringent crystal (with such optical properties). In the fourth embodiment with the mirror box, apertures  270 ,  274  are through-holes of the mirror box. 
     The bottom surface  272  is transparent to the scattered radiation  64 . In the case of the base body made of glass, the bottom surface  272  is uncoated. In the case of the metallic base body, the bottom surface  272  includes a plane-parallel window. In the fourth embodiment with the mirror box, the mirror box is open on the bottom surface  272 . 
     The fifth embodiment of the specimen vessel  256  shown in  FIG. 2  for fluorescent radiation by way of scattered radiation includes a transparent base body which exhibits a dichroic layer on the side surfaces. Alternatively or in supplement, the side surfaces of the specimen vessel consist of a dichroic filter or an interference filter. In this way, the side surfaces of the specimen vessel  256  have been designed to transmit the exciting radiation (of the pencil beam  38 ) and to reflect the fluorescent radiation. In this way, a proportion of the scattered radiation  64  of about 50% or more can be acquired. With regard to the fluorescent radiation (i.e. the scattered radiation  64 ), the side faces act as reflecting surface  269 . Since the side faces are transparent with regard to the exciting radiation (of the pencil beam  38 ), the side surfaces in addition enable the entrance and exit of the pencil beam  38 . That is to say, the entire side surfaces are available by way of illumination aperture  270  or transmission aperture  274 . The control element  30  has been designed for rasterisation (i.e. offset) of the pencil beam in the x-direction and in the y-direction. Without radiation losses, windows (in particular, slits) can be dispensed with. A transportability of the specimen holder  14  in the y-direction is not required. 
     In all the embodiments of the specimen vessel  256 , by reflection (or total reflection) of the scattered radiation  64  on the reflecting surface  269  the scattered radiation  64  is guided to the bottom surface  272 . With a view to integral acquisition of the scattered radiation  64 , a portion of the scattered radiation  272   b  emerging through the bottom surface  272  is acquired. The bottom surface  272  corresponds (functionally) to the measuring aperture  72 . The proportion of the scattered radiation emerging through the bottom surface  272  with respect to the total scattered radiation can be determined as described above in connection with the measuring aperture  72 . Furthermore, the proportion of the scattered radiation emerging through the bottom surface  272  can be determined by a reflection angle at which the scattered radiation is reflected from the reflecting surface  269 . To the extent that the reflection at the reflecting surfaces  269  includes total reflection (as in the first and fifth embodiments of the specimen vessel  256 ), the angle of reflection for determining the proportion is a total reflection angle at the reflecting surface  269 . 
       FIG. 2   b  shows, furthermore, an optional second detector unit  16 ′ which has been described in more detail below in connection with a fourth exemplary embodiment of an apparatus  410 . With a view to enlarging the acquired solid angle, the detector units  16 ,  16 ′ of the various exemplary embodiments can be combined. 
     In order (with any of the embodiments of the specimen vessel  256 ) to couple more than 12.5% of the scattered radiation out from the specimen vessel  256 , the first collimator  76  of the scattered-radiation collector  266  is in direct (refractive-index-adapted) optical contact with the bottom surface  272 . As described above, the first collimator  76  includes a tapered light guide  76   a  on the specimen side. 
     All the further features of the second exemplary embodiment correspond to those of the first exemplary embodiment. In particular, mutually corresponding assemblies  12 ,  14 ,  16  and the components thereof have the same degrees of freedom. 
     The following processes are capable of being implemented with the apparatus  210  which has been described: FLIM (fluorescence-lifetime imaging microscopy); MPM (multi-photon microscopy); FRET (fluorescence resonance energy transfer); FRAP (fluorescence recovery after photobleaching); STED (stimulated emission depletion), whereby after the pencil beam  38  by way of exciting beam a “tube beam” by way of exclusion beam with substantially cylindrically symmetrical intensity distribution is radiated in; FLIP (fluorescence loss in photobleaching); so-called superresolution microscopy; scattered-light microscopy; possibly the following may also be capable of being implemented: STORM (stochastic optical reconstruction microscopy) and FCS (fluorescence correlation spectroscopy). 
       FIG. 3  shows a third exemplary embodiment of an apparatus  310  for tomographic image acquisition. For components of the third exemplary embodiment having the same reference symbols as in the first or second exemplary embodiment, what was described above applies correspondingly, particularly with regard to their properties, constitution, function and their interaction. 
     The third exemplary embodiment differs from one of the preceding exemplary embodiments in that the scattered radiation  64  is collected (i.e. integrated) directly with a first collimator. In other words, with a view to integral acquisition of the scattered radiation  64 , a scattered-radiation collector  366  does not include a light guide  76   a . The scattered-radiation collector  366  includes by way of first collimator a lens  76   b , as described above. In addition, the scattered-radiation collector  366  includes a reflector  390 . The specimen holder  14  and the specimen vessel  56  have been arranged between the reflector  390  and the lens  76   b . The specimen holder  14 , more precisely, the axis of rotation, is situated in a focal plane of the lens  76   b . By way of reflector  390 , a spherical concave mirror or a plurality of retroreflectors (also designated as “cat&#39;s-eye”) has/have been employed. In the latter case, the apparatus  310  is particularly easy to adjust. In the case of the spherical concave mirror, a sphere midpoint is situated in the specimen holder  14 , more precisely on the axis of rotation. In this way, the reflector  390  reflects back (in both cases) the scattered radiation  64  captured in the solid angle of the reflector  390  to its origin. In this way, up to twice as many photons of the scattered radiation  64  can be acquired. For an embodiment in which the reflected scattered radiation  64  (in the form of an almost parallel bundle of rays) goes past the specimen vessel  56 , reference is made to the exemplary embodiment of the apparatus  510  described below. 
     Alternatively, for a comparable increase in sensitivity or reduction in intensity of the incident pencil beam  38 , instead of a reflector  390  two or more reflector segments may also have been arranged around the specimen holder  14 . 
     All the further features of the third exemplary embodiment correspond to those of the first or second exemplary embodiment. In particular, mutually corresponding assemblies  12 ,  14 ,  16  and the components thereof have the same degrees of freedom. 
     The following processes are capable of being implemented with the apparatus  310  which has been described: FLIM (fluorescence-lifetime imaging microscopy); MPM (multi-photon microscopy); FRET (fluorescence resonance energy transfer); FRAP (fluorescence recovery after photobleaching); STED (stimulated emission depletion), whereby after the pencil beam  38  by way of exciting beam a “tube beam” by way of exclusion beam with substantially cylindrically symmetrical intensity distribution is radiated in; FLIP (fluorescence loss in photobleaching); so-called superresolution microscopy; scattered-light microscopy; possibly the following may also be capable of being implemented: STORM (stochastic optical reconstruction microscopy) and FCS (fluorescence correlation spectroscopy). 
       FIG. 4  shows a fourth exemplary embodiment of an apparatus  410  for tomographic image acquisition. For components of the fourth exemplary embodiment having the same reference symbols as in one of the preceding exemplary embodiments, what was described above applies correspondingly, particularly with regard to their properties, constitution, function and their interaction. 
     The fourth exemplary embodiment differs from one of the preceding exemplary embodiments in that (instead of a scattered-radiation collector  66 ;  266 ;  366 ) two or more scattered-radiation collectors  366 ,  366 ′ (or, likewise,  66 ;  266 ) have been arranged around the specimen holder  14 . In other words, the detector unit  16  includes two or more scattered-radiation collectors  366 ,  366 ′ (or, likewise,  66 ;  266 ). 
     In exemplary embodiments (not shown) with a single signal generator  86 , prisms or mirrors have been arranged for the purpose of merging differing beam paths of the collimated scattered radiation  78 ,  78 ′ of each of the two or more scattered-radiation collectors  366 ,  366 ′. The merged collimated scattered radiation is then projected, as described above, onto the single signal generator  86  for the purpose of enlarging an acquired solid angle. 
     In the exemplary embodiment shown in  FIG. 4 , a filter-option unit  80 ;  80 ′, a second collimator  84 ,  84 ′, and a signal generator  86 ;  86 ′ have each been assigned to each of the two or more scattered-radiation collectors  366 ,  366 ′ (or, likewise,  66 ;  266 ). In a variant, a common filter option has been synchronised by means of the filter-option units  80 ,  80 ′, and subsignals of the signal generators  86 ,  86 ′ are added up into one signal for the purpose of enlarging the acquired solid angle. In an alternative variant, the filter option by means of the filter-option units  80 ,  80 ′ is independent, and the two or more signals of the signal generators  86 ,  86 ′ are acquired synchronously. In this way, several signals that correspond to differing colour components in the scattered radiation  64 ,  64 ′ can be acquired simultaneously. This enables a fast acquisition (and reconstruction) of functionally marked colour images, particularly in the case of fluorescent radiation. 
     All the further features of the fourth exemplary embodiment correspond to those of the preceding exemplary embodiments In particular, mutually corresponding assemblies  12 ,  14 ,  16  and the components thereof have the same degrees of freedom. 
     The following processes are capable of being implemented with the apparatus  410  which has been described: FLIM (fluorescence-lifetime imaging microscopy); MPM (multi-photon microscopy); FRET (fluorescence resonance energy transfer); FRAP (fluorescence recovery after photobleaching); STED (stimulated emission depletion), whereby after the pencil beam  38  by way of exciting beam a “tube beam” by way of exclusion beam with substantially cylindrically symmetrical intensity distribution is radiated in; FLIP (fluorescence loss in photobleaching); so-called superresolution microscopy; scattered-light microscopy; possibly the following may also be capable of being implemented: STORM (stochastic optical reconstruction microscopy) and FCS (fluorescence correlation spectroscopy). 
       FIG. 5  shows a fifth exemplary embodiment of an apparatus  510  for tomographic image acquisition. For components of the fifth exemplary embodiment having the same reference symbols as in one of the preceding exemplary embodiments, what was described above applies correspondingly, particularly with regard to their properties, constitution, function and their interaction. 
     The fifth exemplary embodiment differs from one of the preceding exemplary embodiments in that the scattered radiation  64  arises directly in the first collimator  76 , particularly in one of its components  76   a ,  76   b . In other words, the first collimator  76  includes (functionally) the specimen vessel  56 . 
     In the exemplary embodiment shown in  FIG. 5 , a first collimator  576  includes a glass body  576   a  with (by approximation) the shape of an elongated half ellipsoid of revolution. The first collimator  576  exhibits at its rounded end, over about one third of its axial length, a reflector  590 . The reflector  590  can be realised as a reflecting coating on the glass body  576   a , or includes the plurality of retroreflectors (described in connection with reflector  390 ). 
     Still within this third, for instance at one quarter of the axial length, an elongated cuboid collimator recess  556  has been provided. The longitudinal axis of the collimator recess  556  coincides with the (vertical) axis of rotation of the apparatus  510  and intersects the axis of symmetry  594  of the first collimator  576  perpendicularly. The collimator recess  556  extends almost completely over the cross section of the first collimator  576 , so that the collimator recess  556  is closed on one side (at the bottom). The collimator recess  556  is dimensioned in such a way that the specimen holder  14  arranged in the collimator recess  556  (over a length enclosing the specimen) is rotatable in non-contacting manner. In this way, the collimator recess  556  acts as the specimen vessel  56  as described above. In particular, the volume between the specimen holder  14  and the collimator recess  556  contains the refractive-index-adapted liquid  58 . 
     To the extent that a non-cuboid collimator recess  556  does not already exhibit plane side surfaces for entrance and exit of the pencil beam  38 , appropriate plane windows have been provided in the side surfaces. The pencil beam  38  is focused into the specimen holder by the plane entrance window. 
     At the end of the first collimator  576  situated opposite the reflector  590 , a lens  576   b  has been arranged on the axis of symmetry  594  in a conical recess. The lens  576   b  is designed to be similar to the lens  76   b  described above. 
     The optical elements  576   a ,  590  and  576   b  have been arranged to direct almost the entire scattered radiation  64  emerging from the specimen holder  14  with slight residual divergence in the direction of the signal generator  86  of the detector unit  16 . The scattered radiation reflected back by the reflector  590  into the specimen holder  14  is collimated, together with the directly acquired scattered radiation  64 , by the first collimator  576  to such an extent that the acquired radiation can be filtered and detected as described above. 
     All the further features of the fifth exemplary embodiment correspond to those of the preceding exemplary embodiments. In particular, mutually corresponding assemblies  12 ,  14 ,  16  and the components thereof have the same degrees of freedom. 
     The following processes are capable of being implemented with the apparatus  510  which has been described: FLIM (fluorescence-lifetime imaging microscopy); MPM (multi-photon microscopy); FRET (fluorescence resonance energy transfer); FRAP (fluorescence recovery after photobleaching); STED (stimulated emission depletion), whereby after the pencil beam  38  by way of exciting beam a “tube beam” by way of exclusion beam with substantially cylindrically symmetrical intensity distribution is radiated in; FLIP (fluorescence loss in photobleaching); so-called superresolution microscopy; scattered-light microscopy; possibly the following may also be capable of being implemented: STORM (stochastic optical reconstruction microscopy) and FCS (fluorescence correlation spectroscopy). 
     In all the exemplary embodiments with only one x-control element  42  a photosensitive surface of the signal generator  86  (in the y-direction) is capable of being made considerably smaller. As described above, this is also possible in the case of variable y-offset by virtue of a synchronous transporting in the y-direction either of the specimen holder  14  and the specimen vessel  56 ;  256 , or of the light-source  12  and the detector unit  16 . 
       FIG. 6  shows a simplified flow chart of a process  600  for tomographic image acquisition. The process  600  is capable of being implemented by means of any of the exemplary embodiments, described above, of the apparatus  10 ;  210 ;  310 ;  410 ;  510  for tomographic image acquisition. 
     The process  600  for tomographic image acquisition comprises the following steps:
         radiating  602  a pencil beam  38  with a beam direction  40  into a (rotatable) specimen holder  14  with a view to the excitation of scattered radiation  64 ,   non-spatially-resolved acquiring  604  of the scattered radiation  64  excited along a section of the pencil beam  38 ,   substantially parallel offsetting  606  of the pencil beam  38  at right angles to the beam direction  40  of the pencil beam  38  to a plurality of measuring positions with a fast-rasterisation direction and with a slow-rasterisation direction,   rotating  608  the specimen holder  14  to a plurality of rotary positions for each of the plurality of measuring positions, and   tomographic reconstructing  610  of an image (in particular, a sectional image or a spatial image) on the basis (of an intensity) of the acquired scattered radiation  64 .       

     The fast-rasterisation direction is preferentially perpendicular to the slow-rasterisation direction. The slow-rasterisation direction may coincide with the axis of rotation. In connection with one of the apparatuses described above, the x-direction may be the fast-rasterisation direction and/or the y-direction may be the slow-rasterisation direction. The offsetting into the measuring positions is also designated as rasterising. 
     The offsetting can be undertaken by means of the optical control element  30  described above. The pencil beam  38  can be offset in an xy-plane (perpendicular to the beam direction  40 ) in accordance with a “meandering” contour. Alternatively, the offsetting may exhibit a spiral course or may be undertaken along concentric circles. 
     The slow-rasterisation direction and the rotation are interchangeable in the process  600 . That is to say, (at least) the motion of the slow-rasterisation direction and the rotating commute. This may enable a minimisation of the total acquisition time. 
     To the extent that the axis of rotation is parallel to the y-direction, the reconstruction can begin already prior to conclusion of a complete image acquisition. Advantageously, after a partial acquisition with respect to the fast-rasterisation direction and with respect to the rotation (i.e. when the rotation occurs “before” the slow-rasterisation direction) has been concluded, an intersecting plane (perpendicular to the slow-rasterisation direction) is reconstructed. In this way, after each complete rotation a partial reconstruction can begin. 
     A control process (for acquiring the scattered radiation  64 ) may comprise the offsetting in the fast-rasterisation direction by way of “innermost iteration loop”, the rotating by way of “middle iteration loop”, and the offsetting in the slow-rasterisation direction by way of “outer iteration loop”. The partial reconstruction can begin after conclusion of each “middle iteration loop”. Each partial reconstruction is capable of being put into effect independently of a further partial acquisition and independently of a further partial reconstruction. In this way, the partial reconstructions can be effectively parallelised. 
     As has become evident from the description of various exemplary embodiments, the apparatus  10 ;  210 ;  310 ;  410 ;  510  and the process  600  enable a tomographic image acquisition of an at least partly transparent specimen which has been suspended in a refractive-index-matching substance. The radiation-source  18  may also include several lasers, the partial raw beams of which have been superimposed on the raw beam  20 . In this way, a spectrum of the pencil beam  38  can be realised that has been adapted to several transitions of the fluorophores introduced into the specimen (i.e. several colours) 
     The technique that has been described can, depending on the exemplary embodiment, offer one or more of the following advantages: ring artefacts, which may arise in the course of optical projection tomography (OPT), are avoided. The sensitivity in respect of fluorescence (in the case of specimens having a dimension of more than one millimeter) is significantly greater than with known techniques. Consequently, a higher specimen throughput is possible, and there is less potential for damage to the specimen. The apparatus  10 ;  210 ;  310 ;  410 ;  510  requires only components that are available at reasonable cost. 
     LIST OF REFERENCE SYMBOLS 
     (The last two numerals denote the functional feature. In the case of three-digit reference symbols, the leading numeral corresponds to the Figure.)
       10 ,  210 ,  310 ,  410 ,  510  apparatus for tomographic image acquisition     12  light-source     14  specimen holder     16  detector unit     18  radiation-source     20  raw beam     22  beam-shaping lens system     24  telescopic lens system     26  diaphragms and phase masks     28  shaped beam     30  optical control element     32  beam-splitter     34  partial beam     36  reference detector     38  pencil beam     40  beam direction     42  x-control element     44  x-actuator     46  x-rasterisation mirror     48  y-control element     50  y-actuator     52  y-rasterisation mirror     54  (first) focusing lens system     56 ,  256 ,  556  specimen vessel; collimator recess     58  liquid     59  glass surface     60  second focusing lens system     62  transmission photodetector     64 ,  64 ′ scattered radiation     66 ,  266 ,  366 ,  366 ′ scattered-radiation collector     68  integrator     69 ,  269  inner surface, reflecting surface     70 ,  27  illumination aperture     72 ,  272  measuring aperture; transparent bottom aperture     74 ,  274  transmission aperture     76 ,  576  first collimator     576   a  glass body     76   a ,  576   a  light guide, in partic. optical hollow-core conductor     76   b ,  576   b  lens     78 ′ partly collimated scattered radiation     78  collimated scattered radiation     80 ,  80 ′ filter-option unit     82  filtered scattered radiation     84  second collimator     86 ,  86 ′ signal generator     88  stepper motor     390 ,  590  reflector     594  axis of symmetry