Abstract:
A scanning microscope having a light source that emits illuminating light for illumination of a specimen, having at least one first detector for detection of the detected light proceeding from the specimen, having an objective being arranged in both an illumination beam path and a detection beam path, and having a coupling-out element that is selectably for descan detection and non-descan detection positionable in the illumination and detection beam path, is disclosed.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This invention claims priority of the German patent application 101 20 424.8-42 which is incorporated by reference herein. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The invention concerns a scanning microscope having a light source that emits illuminating light for illumination of a specimen, having at least one first detector for detection of the detected light proceeding from the specimen, and having an objective by means of which the specimen can be illuminated and detected, the objective being arranged in both an illumination beam path and a detection beam path.  
           [0003]    The invention further concerns a coupling-out element for a scanning microscope.  
         BACKGROUND OF THE INVENTION  
         [0004]    In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light (in the form of reflected or fluorescent light) emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the current mirror position.  
           [0005]    In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.  
           [0006]    A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detection of the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels via the beam deflection device back to the beam splitter, passes through the latter and is then focused onto the detection pinhole, behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not originate directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which, by sequential scanning of the specimen with the focus of the illuminating light beam, results in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes usually comprise a scan module that is flange-mounted onto the stand of a conventional light microscope and contains all the aforesaid elements additionally necessary for scanning a specimen.  
           [0007]    Commercial scanning microscopes usually contain a microscope stand like the one also used in conventional light microscopy. As a rule, confocal scanning microscopes in particular are also usable as conventional light microscopes. In conventional fluorescent incident-light microscopy, the portion of the light of a light source (for example of an arc lamp) that comprises the desired wavelength region for fluorescent excitation is coupled into the microscope beam path with the aid of a color filter called the excitation filter. Coupling into the beam path of the microscope is accomplished using a dichroic beam splitter, which reflects the excitation light to the specimen while allowing the fluorescent light proceeding from the specimen to pass largely unimpeded. The excitation light scattered back from the specimen is held back with a blocking filter that is, however, transparent to the fluorescent radiation. Combining mutually matched filters and beam splitters optimally to yield an easily interchangeable modular filter block has been common for some time. The filter blocks are usually arranged in a turret within the microscope as a part of so-called fluorescent incident-light illuminators, thus enabling rapid and easy interchanging.  
           [0008]    In confocal scanning microscopy, a detection pinhole can be dispensed in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light being detected therefore very probably originates almost exclusively from the focus region, which renders superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.  
           [0009]    Especially given that the yield of fluorescent photons in two-photon excitation is in any case low, a non-descan arrangement, in which the detected light does not arrive at the detector via the beam deflection device (descan arrangement) and the beam splitter for coupling in the illuminating light, but rather is deflected directly after the objective by means of a dichroic beam splitter and detected, is of interest because less light is generally lost when the detected light is guided in this fashion. In addition, when descan detection is used in two-photon excitation, scattered components of the detected light contribute significantly to the signal, whereas with non-descan detection they play only a greatly reduced role. Arrangements of this kind are known, for example, from the publication by David W. Piston et al., “Two-photon excitation fluorescence imaging of three-dimensional calcium ion activity,” Applied Optics, Vol. 33, No. 4, February 1996, and from Piston et al., “Time-Resolved Fluorescence Imaging and Background Rejection by Two-Photon Excitation in Laser Scanning Microscopy,” SPIE Vol. 1640.  
           [0010]    One problem with the known arrangements is that of arranging a beam splitter, to deflect the detected light out of the microscope beam path after the objective, within a scanning microscope for non-descan deflection, and aligning it precisely. This requires the implementation of complex additional arrangements that necessitate massive physical modifications to the scanning microscope and in particular to the microscope stand. Retrofitting to a scanning microscope with descan detection is usually impossible or very complex.  
         SUMMARY OF THE INVENTION  
         [0011]    It is therefore the object of the invention to propose a scanning microscope which is operable selectably with descan detection or with non-descan detection and with which it is possible to switch over easily and reliably between descan detection and non-descan detection.  
           [0012]    The aforesaid object is achieved by means of a scanning microscope comprising:  
           [0013]    a light source for emitting illuminating light for illumination of a specimen ( 19 ),  
           [0014]    at least one first detector for descan detection of the detection light proceeding from the specimen,  
           [0015]    an objective being arranged in both an illumination beam path and a detection beam path,  
           [0016]    a coupling-out element being insertable into the illumination and detection beam path for non-descan detection, and removable from the illumination and detection beam path for descan detection  
           [0017]    A further object of the invention is to disclose a coupling-out element which easily enables a scanning microscope to be operated selectably with descan detection or with non-descan detection.  
           [0018]    This object is achieved by means of a coupling-out element for a scanning microscope, which is insertable into an illumination and detection beam path of the scanning microscope for non-descan detection, and which is removable from the illumination and detection beam path for descan detection.  
           [0019]    The invention has the advantage of making it possible to switch over easily between descan detection and non-descan detection.  
           [0020]    In a preferred embodiment, the coupling-out element contains a beam splitter that is preferably configured as a dichroic beam splitter or color beam splitter. The beam splitter is preferably configured to be transparent to the illuminating light and reflective to the detected light.  
           [0021]    In a further preferred embodiment, the coupling-out element comprises filters that act as excitation filters on the light in the illumination beam path or as detection filters on the light in the detection beam path. In a very particularly preferred embodiment, the coupling-out element contains both an excitation filter and a detection filter. The detection filter is preferably configured in such a way that it allows only the detected light proceeding from the specimen, and in particular no light of the wavelength of the illuminating light, to pass, thus advantageously preventing any undesirable and falsifying detection of that light in particular. The excitation filter serves to filter out from the spectrum of the light source those light components having the wavelengths of interest for illumination.  
           [0022]    In a variant embodiment, the coupling-out element can be introduced from outside into the illumination and detection beam path in order to switch over from descan detection to non-descan detection, guidance elements such as guide rails, slide bars, or a bayonet mount, which make possible simple and reliable introduction and positioning, being provided. Also provided are banking elements, which define a working position of the coupling-out element in the illumination and detection beam path and which are configured so that the positioned coupling-out element is automatically aligned with respect to the detection beam path, and no further alignment of the coupling-out element is necessary after positioning.  
           [0023]    In another preferred embodiment, a turret or a sliding carriage which comprises at least one element receptacle is provided for positioning the coupling-out element, the coupling-out element being mounted on or in the element receptacle in such a way that the coupling-out element can be positioned in the illumination and detection beam path by simply rotating the turret or sliding the sliding carriage. Alignment of the coupling-out element is performed only once, when the coupling-out element is mounted in or on the turret or sliding carriage. The latter advantageously has a snap-in apparatus that releasably immobilizes the turret or sliding carriage when the coupling-out element is positioned in the illumination and detection beam path. In a further variant embodiment, the turret or sliding carriage comprises several element receptacles in which other optical elements, for example filters or additional optical systems, are mounted. The turret or sliding carriage also comprises at least one open position that can be introduced into the illumination and detection beam path in such a way that the illuminating light and detected light can pass unimpeded. This manner of achieving the object of the invention is economical and highly flexible, since several coupling-out elements having different beam splitters with different spectral properties can be held in readiness and easily interchanged.  
           [0024]    In a very particularly preferred embodiment, the coupling-out element is a component of a fluorescent incident-light illuminator. This embodiment is very particularly advantageous in scanning microscopes that are also suitable for conventional incident-light microscopy, which contain a fluorescent incident-light illuminator with a turret. This has the advantage of utilizing or putting to a different purpose apparatuses which usually are present in any case, thereby avoiding any physical modification to the microscope or the microscope stand. By simply rotating the turret of the fluorescent incident-light illuminator, it is possible to switch between conventional fluorescence microscopy, descan detection, and non-descan detection.  
           [0025]    The coupling-out element delivers at least a portion of the detected light to a further detector. In an embodiment, this detector is mounted externally on the scanning microscope, the scanning microscope having an opening in the housing through which, in the context of non-descan detection, the detected light emerges and arrives at the further detector. In another embodiment, the further detector is a component of the coupling-out element. Semiconductor detectors, such as photodiodes or avalanche photodiodes, are particularly advantageous in this context because of their small overall size. Photomultipliers are also usable. In a particularly preferred embodiment, the further detector contains at least two further individual detectors for separate detection of different spectral regions of the detected light. Beam splitters, which advantageously are housed in a filter block, are provided in order to divide the detected light. Further filters and/or optical systems can be inserted into the filter block. It is very particularly advantageous, especially in terms of integrated manufacture, if the filter block has the same dimensions and guidance and/or banking elements as the coupling-out element.  
           [0026]    In a particular variant embodiment, in non-descan detection mode the detected light coupled out by means of the coupling-out element is delivered to the detector actually provided for descan detection. Mirror arrangements or light-guiding fibers are provided for this purpose. This variant embodiment has the particular advantage that a single detector alone can be used for both descan detection and non-descan detection, which makes the entire scanning microscope much simpler and less expensive to manufacture. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0027]    The subject matter of the invention is schematically depicted in the drawings and will be described below with reference to the Figures, identically functioning elements being labeled with the same reference characters. In the drawings:  
         [0028]    [0028]FIG. 1 shows a known arrangement for fluorescence microscopy, having several interchangeable modular filter blocks in a turret;  
         [0029]    [0029]FIG. 2 is a plan view of the arrangement from FIG. 2;  
         [0030]    [0030]FIG. 3 shows a scanning microscope according to the present invention;  
         [0031]    [0031]FIG. 4 is a plan view of the scanning microscope according to the present invention from FIG. 4;  
         [0032]    [0032]FIG. 5 shows a further scanning microscope according to the present invention;  
         [0033]    [0033]FIG. 6 shows a further scanning microscope according to the present invention;  
         [0034]    [0034]FIG. 7 shows a coupling-out element; and  
         [0035]    [0035]FIG. 8 shows a further coupling-out element. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0036]    [0036]FIG. 1 schematically shows an incident-light fluorescence microscope  1  known from the existing art, having a fluorescent incident-light illuminator  3 . Excitation filter  11  is used to filter out from light  9  coming from light source  5 , which is embodied as an arc lamp  7 , those components having the desired wavelengths (i.e. excitation light  13 ). Excitation light  13  is then reflected from dichroic beam splitter  15  toward microscope objective  17 , which focuses excitation light  13  onto specimen  19 . Fluorescent light  21  proceeding from the specimen travels back through the microscope objective to dichroic beam splitter  15 , passes through it, and is incident through blocking filter  23  and eyepiece  25  into the user&#39;s eye  27 . Excitation filter  11 , blocking filter  23 , and dichroic beam splitter  15  are arranged in an interchangeable modular first filter block  29 .  
         [0037]    First filter block  29  is arranged, together with a further filter block  31  that contains an excitation filter  33 , a detection filter  35 , and a beam splitter  37 , in a turret  41  that can rotate about shaft  39 . Excitation filter  33 , detection filter  35 , and beam splitter  37  have different spectral properties from excitation filter  11 , detection filter  23 , and beam splitter  15  of first filter block  29 . Filter block  29 ,  31  having the desired optical properties can be brought into the microscope&#39;s beam path by rotating the turret.  
         [0038]    [0038]FIG. 2 shows incident-light fluorescence microscope  1  in a plan view, in which it is evident that the turret contains not only filter blocks  29  and  31  but also two further filter blocks  43 ,  45 .  
         [0039]    [0039]FIG. 3 shows a scanning microscope  47  according to the present invention. Illuminating light  53  coming from a light source  49 , which is embodied as a mode-locked titanium-sapphire laser  51 , has a wavelength of approx. 800 nm and is focused by optical system  55  onto excitation pinhole  57 , and is then reflected by a beam splitter  59  to beam deflection device  61  which contains a gimbal-mounted mirror  63 . Scanning optical system  65 , tube optical system  67 , and objective  69  define an illumination beam path  71  and a detection beam path  72 , along which illuminating light  53 , shaped into a beam, is guided over or through specimen  19 . Located between tube optical system  67  and objective  69  is coupling-out element  73 , containing a dichroic beam splitter  77  and an excitation filter  74 , for coupling out detected light  75  proceeding from the sample. Dichroic beam splitter  77  is configured so that illuminating light  53  having a wavelength of approx. 800 nm can pass unimpeded, and detected light  75  is reflected (out of the plane of the drawing) toward further detectors  80 ,  81  (not shown in this Figure). Coupling-out element  73  is arranged in a turret  85  that can rotate about shaft  83 , and is aligned in such a way that by rotation of turret  85 , it can be positioned in the excitation and detection beam path. Arranged in turret  85  is a further coupling-out element  87  that contains an excitation filter  89 , a detection filter  91 , and a beam splitter  93 , these elements having spectral properties different from those of first coupling-out element  73 . The coupling-out element  73 ,  87  that has the particular desired optical properties can be positioned in illumination and detection beam path  71 ,  72  by rotation of turret  85 . For descan detection, turret  85  can additionally be rotated into an open position  99 ,  101  (not depicted in this Figure) so that both illuminating light  53  and detected light  75  pass unimpeded. In descan detection mode, detected light  75  travels via beam deflection device  61  back to beam splitter  59 , passes through the latter, through blocking filter  79  which suppresses residual radiation from the excitation light, and through detection pinhole  95 , and then strikes first detector  97 .  
         [0040]    [0040]FIG. 4 shows a plan view of the scanning microscope depicted in FIG. 3. Illuminating light  53  is incident onto the plane of the drawing. Detected light  75  emerges laterally from coupling-out element  73  and encounters filter block  104 , which contains a dichroic beam splitter  103  and two blocking filters  106 ,  108 . At dichroic beam splitter  103 , which is embodied as a color beam splitter, detected light  75  is divided in accordance with the spectral distribution into beam segments  105  and  107  and conveyed to detectors  80  and  81 , which are embodied as photomultipliers. The two blocking filters  106 ,  108  are configured so that only detected light of the respectively desired wavelength reaches detectors  80 ,  81 . Open positions  99 ,  101  for descan detection are drawn with dotted lines.  
         [0041]    [0041]FIG. 5 shows, in a perspective view, a confocal scanning microscope  47  that comprises a conventional light microscope  109  and a scanner module  111 . Scanner module  111  contains a light source  49  for generating illuminating light  53 , a beam deflection device  61 , a first detector  97  for descan detection, and a beam splitter  59  that reflects illuminating light  53  to beam deflection device  61  and, in descan detection mode, allows the detected light to pass to first detector  97 . Scanner module  111  furthermore contains elements (not shown) for beam guidance and shaping, as well as an excitation pinhole and a detection pinhole, which in the interest of clarity also are not shown. Specimen  19  rests, together with a specimen slide  113 , on a microscope stage (not shown). Conventional light microscope  109  comprises a housing (not depicted) onto which scanner module  111  is flange-mounted. Conventional light microscope  109  furthermore contains a fluorescent incident-light illuminator  3  having a light source  5  that is embodied as an arc lamp  7 , and a turret  41  that is rotatable about shaft  39 . A coupling-out element  73  for scanning microscopy with non-descan detection, and a first and a second filter block  29 ,  31  for conventional fluorescent incident-light microscopy, are arranged in the turret. Turret  41  also comprises an open position  99  that can be rotated into illumination and detection beam path  71 ,  72 . This position is used for scanning microscopy with descan detection. The housing of conventional light microscope  109  has a lateral opening through which, in non-descan detection mode, detected light  75  emerges and travels to further detectors  80  and  81  that are mounted on the housing.  
         [0042]    [0042]FIG. 6 shows a scanning microscope  47  according to the present invention which is constructed for the most part exactly like scanning microscope  47  shown in FIG. 5. What is used for non-descan detection in this embodiment, however, is not further detectors  80 ,  81  but rather first detector  97  that is actually provided for descan detection. Detected light  75  is delivered to the latter via a light-guiding fiber  115  and a mirror  117 , bypassing the detection pinhole.  
         [0043]    [0043]FIG. 7 shows a coupling-out element  73  according to the present invention that contains an excitation filter  7 , a detection filter  119 , and a dichroic beam splitter  77 . Coupling-out element  73  comprises a housing  121  having three openings  123 ,  125 ,  127  through which illuminating light  53  and detected light  75  pass. Mounted on housing  121  are guidance and/or banking elements  129 ,  131 ,  133 ,  135  which make possible simple and reproducible positioning in excitation and detection beam path  71 ,  72 .  
         [0044]    [0044]FIG. 8 shows a coupling-out element  73  according to the present invention having an integrated further detector  137  that is configured as a photodiode. An optical system  120  is arranged in front of the detector.  
         [0045]    The present invention has been described with reference to a particular embodiment. It is nevertheless self-evident that changes and modifications can be made without thereby leaving the range of protection of the following claims.