Abstract:
The invention relates to a laser scanning microscope with a scanner and a microscope objective, and to a control method for such a microscope. In order to obtain sharp imaging of the sample in a laser scanning microscope, the distance between the microscope objective and the sample is usually varied for adjusting the focus position. However, relative movements between the objective and the sample can be problematic. In view of the costly special objective, internal focusing of the objective is a disadvantageous solution. An improved laser scanning microscope should make it possible to sharply image a sample with standard objectives without relative movement between the microscope objective and sample. According to the invention, a tube lens is provided which is displaceable along the optical axis, and the focus position is adjustable relative to a front optical element of the microscope objective by adjusting the tube lens.

Description:
[0001]    The present application claims priority from PCT Patent Application No. PCT/EP2008/006457 filed on Aug. 6, 2008, which claims priority from German Patent Application No. 10 2007 038 579.1 filed on Aug. 16, 2007, the disclosure of which is incorporated herein by reference in its entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The invention is directed to a laser scanning microscope and to a control method for a laser scanning microscope of this kind. 
         [0004]    2. Description of Related Art 
         [0005]    In order to obtain sharp imaging of the sample in a laser scanning microscope, the distance between the microscope objective and the sample is usually varied to adjust the focus position. To this end, either the sample stage with the sample or the microscope objective can be moved. It is also possible to combine the two movements. 
         [0006]    However, the relative movements between the objective and the sample are problematic under some circumstances. For example, in electrophysiological experiments, a large number of delicate contacts are connected to the sample. A movement of the sample stage could cause these contacts to be torn out and, for this reason, is generally ruled out for fixing the focus position. Aside from this, movement of the objective may also be ruled out, for example, when contacts are guided past close to the objective or when the objective is immersed in a specimen liquid. 
         [0007]    So-called internal focusing of objectives is known in microscopes in the prior art. For example, DE 10 2005 034 441 A1 shows a microscope objective with an adjusting device by means of which an optical element inside the objective is displaceable along the optical axis in such a way that the focus position is displaced in direction of the optical axis relative to the front element of the objective. 
         [0008]    Sharp imaging can be achieved without relative movement of the objective and sample by means of internal focusing of the objective. However, the required special objective is extremely costly. 
       SUMMARY OF THE INVENTION 
       [0009]    Therefore, it is the object of the invention to provide a laser scanning microscope and a method for controlling the latter which also makes possible a sharp imaging of a sample with standard objectives without relative movement of the microscope objective and sample. 
         [0010]    This object is met by a laser scanning microscope having a scanner, a microscope objective, and a tube lens which is adjustable along an optical axis of the microscope. A focus position of this microscope is adjustable relative to a front optical element of the microscope objective by adjusting the tube lens. 
         [0011]    This object is also met by a control method for the above laser scanning microscope, where an optical section of a sample is scanned by means of the scanner. The focus position is subsequently adjusted by adjusting the tube lens. Then, the process is repeated. In this method, a movement of the scanner is influenced based on a given correction value for an imaging scale of the adjusted focus position in such a way that all of the optical sections of the sample are recorded with approximately the same imaging scale. 
         [0012]    Advantageous embodiments of the invention are indicated in the dependent claims. 
         [0013]    According to the invention, a laser scanning microscope has a scanner, a microscope objective, and a tube lens which is adjustable along the optical axis, the focus position being adjustable relative to a front optical element of the microscope objective by adjusting the tube lens. Within the meaning of the invention, the term adjustable tube lens also includes any adjustable optical system acting in a corresponding manner which is arranged between the microscope objective and the scanner in the microscope beam path. 
         [0014]    Since the tube lens is arranged and can be adjusted outside the microscope objective, the microscope can advantageously be provided with a standard objective. A sharp imaging of the sample can nevertheless be obtained without relative movement between the microscope objective and sample. 
         [0015]    In a preferred embodiment, the laser scanning microscope is outfitted with a control unit which repeatedly scans an optical section of a sample by means of the scanner, and a different focus position can be adjusted subsequently by displacing the tube lens. In this way, a stack of optical sections from different focus distances can advantageously be recorded in a z-scan without relative movement between the microscope objective and sample. In a z-scan of this kind, the control unit preferably determines a plurality of discrete focus positions step by step over a depth range of the sample and, in each focus position, scans a respective optical section as part of a z-stack recording. In this way, a z-stack can be recorded in an automated manner. 
         [0016]    Surprisingly, it was found that a laser scanning microscope with an aperture between 0.45 and 0.53, in particular an aperture of 0.45 or 0.53, and/or with an immersion medium having a refractive index between 1.31 and 1.36 can adjust the focus position in a large depth range of 200 μm. 
         [0017]    A disadvantage of a z-scan by means of internal focusing consists in that the imaging scale changes when the focus position is displaced because the magnification is determined not only by the focal distance ratio between the tube lens and the microscope objective but also by the distance of the respective section plane from the microscope objective and by the distance between the microscope objective and the tube lens. Accordingly, quantitative evaluations of the section recordings, for example, comparisons of data from different sample depths, are problematic. Therefore, when a uniform imaging scale is required, the sections must be scaled differently prior to further use by means of image processing algorithms, which is time-consuming. 
         [0018]    This disadvantage is circumvented with an embodiment form in which the control unit influences a movement of the scanner based on a given correction value for an imaging scale of the adjusted focus position in such a way that all of the optical sections can be recorded with approximately the same imaging scale. When recording an optical section, for example, the scanners are deflected on one or both scanning axes with increasing depth of the focus position with changed amplitude in order to compensate for the change in imaging scale. A lookup table with correction values for different focus positions which can be calculated beforehand or determined in calibration passes is preferably stored in the control unit. Intermediate values can then be determined, for example, by interpolation. Different correction values can be provided for both scan axes for the same focus distance. A set of a plurality of correction values can also be provided for a correction function for every focus position. 
         [0019]    In a further development of this embodiment form, a laser scanning microscope has, in addition to or in place of an adjustable tube lens, an optical element which is displaceable along the optical axis in the microscope objective or collimating optics which are displaceable along the optical axis and by means of which the focus position can be adjusted by the control unit. The advantageous inventive correction of the varying imaging scale can also be used with more complex arrangements. 
         [0020]    An intermediate image plane between the microscope objective and a scanning objective advisably has a constant position relative to the scanning objective, in particular also relative to the microscope objective. 
         [0021]    The control method according to the invention is preferably carried out by the above-mentioned control unit. Alternatively, the control method may also be implemented in some other way. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0022]      FIG. 1  shows a schematic view of a microscope with an adjustable tube lens; 
           [0023]      FIG. 2  shows a section from the beam path of the microscope for two different positions of the tube lens; 
           [0024]      FIG. 3  shows the same section from the beam path of the microscope with additional correction of the scanning movement; and 
           [0025]      FIG. 4  shows a flowchart showing the control method. 
       
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
       [0026]    It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements which are conventional in this art. Those of ordinary skill in the art will recognize that other elements are desirable for implementing the present invention. However, because such elements are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements is not provided herein. 
         [0027]    The present invention will now be described in detail on the basis of exemplary embodiments. 
         [0028]    Corresponding parts have identical reference numbers in all of the drawings. 
         [0029]      FIG. 1  shows a microscope  100  schematically. It comprises a microscope unit M and a scan head S which share a common optical interface by means of an intermediate imaging in an intermediate image plane Z. The microscope beam path of the microscope unit M comprises a light source  1 , illumination optics  2 , a beamsplitter  3 , a microscope objective with water immersion (n=1.33), a tube lens  9  which is adjustable along the optical axis, the observation beam path with an ocular tube lens  10  and an eyepiece  11 , and a beamsplitter  12  for coupling in the scanning beam from the scan head S. A sample  5  to be examined is arranged on a sample stage, not shown, of the microscope  100  under the microscope objective. 
         [0030]    Laser modules  13 . 1 ,  13 . 2  provide, for example, four lasers which are connected by light-conducting fibers  14 . 1 ,  14 . 2  to the laser input-coupling unit of the scan head S. Coupling into the light-conducting fibers  14 . 1 ,  14 . 2  is carried out by collimating optics  16  and beam deflecting elements  17 . 1 ,  17 . 2 . A monitoring beam path is reflected out in direction of a monitor diode  19  by means of a semitransparent mirror  18 . Line filters  21  and neutral filters  20  which are advantageously arranged on a rotatable filter wheel, not shown, are disposed in front of this monitor diode  19 . 
         [0031]    The actual scanning unit comprises a scanning objective  22 , scanner  23 , main beamsplitter  24  and shared imaging optics  25  for detection channels  26 . 1 - 26 . 4 . A deflecting prism  27  behind the imaging optics  25  reflects the radiation coming from the sample  5  in direction of dichroic beamsplitters  28  in the convergent beam path of the imaging optics  25 . Pinholes  29  whose diameter can be changed individually for each detection channel  26 . 1 - 26 . 4  and emission filters  30  and suitable receiver elements  31  (in this case, for example, secondary electron multipliers—PMTs) are arranged downstream of the imaging optics  25 . In further embodiments (not shown), the beamsplitters  28  can be constructed as splitter wheels with a plurality of positions which can be switched through mechanically by stepper motors. 
         [0032]    Single-wavelength lasers and multi-wavelength lasers which can be coupled into the fiber  14 . 2  individually or in combination via an AOTF are provided in the laser module  13 . 2 . In alternative embodiment forms (not shown), coupling in can also be carried out via a plurality of fibers in parallel whose radiation is mixed on the microscope side by color combiners after passing through adapting optics. Mixing of the radiation of different lasers at the fiber input is also possible and can be carried out by means of the exchangeable and switchable splitter mirrors  39  which are shown schematically. 
         [0033]    UV radiation is coupled into the glass fiber  14 . 1 , preferably a single-mode glass fiber, by means of an AOTF  32  serving as a beam deflector. If the beam should not impinge on the fiber input, it is deflected from the fiber input (e.g., in direction of a light trap (not shown)) by means of the AOTF  32 . The input-coupling optics  33  for coupling in the laser radiation has lens systems, not shown, whose focal length is determined by the beam cross section of the lasers and the numerical aperture required for optimal input coupling. The laser radiation exiting divergently from the end of the fibers  14 . 1 ,  14 . 2  at the scan unit S is collimated to an infinite beam by the collimating optics  16 . 
         [0034]    A central control unit  34  controls the variable structural component parts such as tube lens  9 , scanner  23 , AOTF  32 , and splitter mirrors  39 . At the same time, it provides the interface for operating controls and display elements (not shown) and enables a user in particular to manipulate the variable structural component parts (e.g., by means of a connected microcomputer (not shown)). The variable structural component parts can also be controlled automatically by the control unit  34  in automatic sequences. 
         [0035]    The distance between the sharply imaged planes within the sample  5  and the microscope objective  4  can be changed by displacing the tube lens  9  along the optical axis without having to move the microscope objective  4  or sample  5 . This makes its possible in particular to record optical sections at different depths. 
         [0036]      FIG. 2  shows the area enclosed by dashed lines in  FIG. 1  without the beamsplitters  12 ,  24 . The effects of a displacement of the tube lens  9  between two positions A and B taken by way of example are shown schematically referring to two (alternative) beam paths SGA, SGB between the scanner  23  and sample  5 . The position of the intermediate image plane Z is identical for all of the positions of the tube lens  9  with respect to the scanning objective  22  and with respect to the microscope objective  4 . The distance between the sample  5  and microscope objective  4  is also constant. The beam path between the tube lens  9  and the microscope objective or sample  5  changes as the position of the tube lens  9  changes. The deviations are shown in an exaggerated manner in order to show the differences between the beam paths A and B more clearly. Accordingly, the opening angle shown in the drawing is not true to scale. In position A of the tube lens  9 , the focus lies in a near-surface plane E 1  of the sample  5 . When the tube lens  9  is displaced into position B, the focus moves into the near-surface plane E 2 . 
         [0037]    This can be made use of to record a z-stack of optical sections. The control unit  34  moves the focus (e.g., in discrete steps) over a given depth range of the sample by adjusting the tube lens  9 . In every discrete focus position, the tube lens  9  is halted for a scanning process and a corresponding section is recorded in sharp imaging and stored. The z-stack can be evaluated as a three-dimensional image in a known manner. 
         [0038]    Because of the increased distance from the microscope objective, the imaging scale of the microscope  100  in position A and in the positions between position A and position B is greater than in position B. This results in different imaging scales within a z-stack. The optical sections of a z-stack can be converted to a uniform imaging scale by software using image processing algorithms. 
         [0039]    Corresponding to  FIG. 2 ,  FIG. 3  shows a beam path section of a special embodiment form of the microscope  100  in which the different imaging scales are compensated already during the recording of a z-stack by influencing the movement of the scanner  23  when recording an optical section. To this end, the control unit  34  is provided with a lookup table  35  in which sets of previously determined correction values for the imaging scales in a plurality of focus distances are stored. 
         [0040]      FIG. 4  shows the sequence of the control method. To record a z-stack of corrected optical sections, an initial focus position which can be selected by a user is first adjusted in the sample by the control unit  34  in step S 1  by displacing the tube lens  9 . 
         [0041]    In step S 2 , the sample is scanned transverse to the optical axis of the microscope  100  in a known manner. In so doing, the regular movement of the scanner  23 , in other words, the scanner mirror (not shown), is manipulated. This is carried out in that the amplitude of the mirror movement is modified based on the correction values stored for the focus distance in question. For example, there is a smaller mirror deflection for a greater focus distance (i.e., a focus position deeper in the sample  5  at which there is a greater imaging scale). Accordingly, the greater imaging scale can be compensated. In further embodiments (not shown), the scanner mirror can also carry out a more complex movement. The necessary correction values can be determined by theoretical optics calculations and/or calibrating measurements. 
         [0042]    Finally, in step S 3  the recorded optical section is stored. In step S 4 , the control unit  34  adjusts the next focus position. If it is determined that the z-stack has not yet been traversed completely, the method advances to step S 2 . 
         [0043]    Otherwise, in step S 5  the z-stack is read out, for example, to a storage medium, and the process is terminated. Alternatively or in addition, the z-stack can be transferred via an interface to a microcomputer for further processing or evaluation. 
         [0044]    While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims. 
       REFERENCE NUMBERS 
       [0000]    
       
           1  light source 
           2  illumination optics 
           3  beam splitter 
           4  microscope objective 
           5  sample 
           9  tube lens 
           10  ocular tube lens 
           11  eyepiece 
           12  beamsplitter 
           13 . 1 ,  13 . 2  lasers 
           14  light-conducting fibers 
           16  collimating optics 
           17 . 1 ,  17 . 2  beam deflecting element 
           18  semitransparent mirror 
           19  monitor diode 
           20  neutral filter 
           21  line filter 
           22  scanning objective 
           23  scanner 
           24  main beamsplitter 
           25  imaging optics 
           26 . 1 - 26 . 4  detection channels 
           27  deflecting prism 
           28  dichroic beamsplitter 
           29  pinhole 
           30  emission filter 
           31  receiver element 
           32  AOTF 
           33  coupling optics 
           34  control unit 
           35  lookup table 
           39  beamsplitter 
         M microscope unit 
         S scan head 
         E 1  near-surface focus plane 
         E 2  near-surface focus plane 
         A, B position of the tube lens 
         SGA beam path in position A of the tube lens 
         SGB beam path in position B of the tube lens 
         Z intermediate image plane