Abstract:
The invention relates to a microscope having a stage for supporting a sample to be examined, a recording sensor, an imaging optic for imaging the sample onto the recording sensor, a moving unit by means of which the distance between the stage and the imaging optic can be changed, a control unit for controlling an image recording of the sample and a focus-holding unit for maintaining a prescribed focal position for image recording of the sample at temporal intervals, wherein the focus-holding device comprises at least one hardware element and one software module, wherein the focus-holding unit is fully integrated in the control unit, on both the hardware and software sides.

Description:
PRIORITY CLAIM 
       [0001]    The present application is a National Phase entry of PCT Application No. PCT/EP2009/002503, filed Apr. 4, 2009, which claims priority from German Application Number 102008018951.0, filed Apr. 15, 2008, the disclosures of which are hereby incorporated by reference herein in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to a microscope with a stage for supporting a sample to be examined, an image sensor, imaging optics for mapping the sample onto the image sensor, a motion unit for adjusting the distance between stage and imaging optics, a control unit for controlling of an image acquisition of the sample. 
       BACKGROUND OF THE INVENTION 
       [0003]    A microscope with a retaining focus unit is utilized particularly for the observation of processes that take place temporally over several hours or even several days in order to ensure that all exposures are taking place in the same focal position and/or imaging position. 
         [0004]    Previously known retaining focus units are designed as a module separate from the microscope and which is subsequently attached and connected to an existing microscope. A separate control panel is usually provided for operation. This causes the handling and/or the use of the retaining focus unit to be elaborate and inconvenient. In particular, the required space increases since an additional control panel is required. 
       SUMMARY 
       [0005]    In an embodiment the invention is a microscope with a stage for supporting a sample to be examined, an image sensor, imaging optics for mapping the sample onto the image sensor, a motion unit for adjusting the distance between stage and imaging optics, a control unit for controlling of an image acquisition of the sample, and a retaining focus unit for maintaining a predetermined focal position for temporally spaced image acquisitions of the sample, whereby the retaining focus unit contains at least one hardware element and one software element. 
         [0006]    Based on the aforementioned, it is a feature and advantage of embodiments of the invention to further develop a microscope of the initially described type in such a way that the retaining focus unit can be provided simply and with minimal required space. 
         [0007]    According to embodiments of the invention, the retaining focus unit, with regard to software as well as hardware, is fully integrated in the control unit. 
         [0008]    In embodiments of the invention, a separate control panel for the retaining focus unit becomes unnecessary. The control and/or the input and output of information for the retaining focus unit can be effected via the output/input unit, usually already present in the microscope. 
         [0009]    Furthermore, in embodiments of the invention, the complete integration of the retaining focus unit in the control unit of the microscope offers a multitude of advantages for the operation of the microscope. 
         [0010]    For example, the retaining focus unit can access all technically relevant data of the microscope and take them into account for the maintaining of the predetermined focal position. This refers particularly to data that characterize the imaging optics, such as magnification, depth of field, the applied wavelengths, etc. 
         [0011]    Through the integration of the retaining focus unit in the control unit of the microscope, according to embodiments of the invention, the retaining focus functionality can be seamlessly integrated in the operating concept of the microscope without requiring an additional control panel and/or special software for the control of the retaining focus functionality. In addition, the retaining focus unit can execute an independent calibration with unknown microscope lenses of the imaging optics and/or determine the magnification of the imaging optics by means of the retaining focus unit. Furthermore, microscope data, which describe technical properties of the microscope, can be prompted by the retaining focus unit and utilized for maintaining the predetermined focal position. The retaining focus unit can, for its part, store the ascertained data for the microscope in a respective memory. 
         [0012]    If the imaging optics exhibit lens systems with chips for the storage of lens data, in embodiments of the invention, the retaining focus unit can read said chips and/or describe said chips. 
         [0013]    Moreover, in embodiments of the invention, the retaining focus unit can determine a cover glass thickness and/or the thickness of the bottom of a vessel in which the sample was placed, such as a Petri dish or a culture vessel. It is also possible to automatically determine a desired focal position by means of the retaining focus unit. 
         [0014]    Particularly, in embodiments of the invention, different modes of operation of the microscope can be implemented by means of the retaining focus unit. Said modes of operation can refer to the determination of the magnification of the imaging optics, determination of the cover glass thickness and/or bottom thickness of a vessel for the sample, and/or the determination of a desired focal plane. 
         [0015]    In embodiments of the invention, the retaining focus unit can control the motion unit for maintaining the predetermined focal position. Particularly, the retaining focus unit can approach a predetermined focal and/or imaging position (e.g., after the stage was greatly displaced in the imaging direction in order to manipulate or change the sample) automatically or upon request by the operator. 
         [0016]    The retaining focus unit can also control additional components of the microscope. It can, for example, execute a lens change if deemed necessary. 
         [0017]    In embodiments of the invention, for maintaining the predetermined focal position, a set object position is maintained optically constant to the imaging optics. In other words, the optical path length is maintained constant. 
         [0018]    The control unit in the microscope, according to embodiments of the invention, can exhibit an assigned memory that the retaining focus unit can access through writing and/or reading. The memory can, particularly, be integrated as a hardware element directly in the control unit  9  or be connected to said unit. The retaining focus unit can, for example, store and/or write in collected, technically relevant microscope data, such as magnification of the currently applied lens system of the imaging optics, in the memory of the control unit  9 . Furthermore, the retaining focus unit can naturally also access the memory of the control unit with a reading function in order to read technically relevant microscope data stored therein and to take into account for the retaining focus control. 
         [0019]    Moreover, in embodiments of the invention, additional components of the microscope can access the memory of the control unit. Thereby, this can, once again, relate to access for writing and/or reading. 
         [0020]    In embodiments of the invention, the microscope can exhibit at least one lens system with a memory that the retaining focus unit can access through writing and/or reading. The memory of the lens system preferably contains data that characterize the lens system and its properties. Of course, the imaging system of the microscope can exhibit several lens systems that can be selectively introduced into the imaging beam path by means of a nosepiece. Said several lens systems can exhibit a memory each, which the retaining focus unit can access individually through writing and/or reading. 
         [0021]    Furthermore, in embodiments of the invention, it is possible for additional components of the microscope to access the memory of the lens system and/or systems. 
         [0022]    The microscope can be designed as a reflected light microscope or a transmitted light microscope, as a confocal microscope and/or a laser-scanning microscope. Moreover, the microscope can be realized as a fluorescence microscope. The microscope can comprise an illumination unit. 
         [0023]    In embodiments of the invention, the retaining focus unit (particularly, the optical realization) can be designed, e.g., as described in WO2007/144197 A1. Of course, a similar design of the retaining focus unit is also possible. The content of WO2007/144197 A1 is herewith incorporated in the application by reference. 
         [0024]    Furthermore, in embodiments of the invention, a microscopy method is provided, whereby, under the control of a control unit, temporally spaced images of a sample to be examined are mapped via imaging optics, which maps the sample onto an image sensor, whereby a retaining focus unit, which contains at least one hardware element and one software module, is provided for maintaining a predetermined focal position for the temporally spaced image acquisitions of the sample, and whereby the retaining focus unit, with regard to software as well as hardware, is also fully integrated in the control unit. 
         [0025]    With the microscopy method, according to embodiments of the invention, the retaining focus functionality can be seamlessly integrated in the operating concept of the control unit, which, overall, leads to an easier to operate microscope. 
         [0026]    With the microscopy method, according to embodiments of the invention, the magnification of the imaging optics can be determined by means of the retaining focus unit. For example, this can be realized in such a way that the retaining focus unit detects the sample to be examined via the imaging optics; thereby, the distance between imaging optics and sample is varied. From the resulting change of the detected signal (for example, a shift of the detected signal in a plane of a focus measurement sensor of the retaining focus unit), the magnification of the imaging optics can subsequently be derived. 
         [0027]    In addition, the method, according to embodiments of the invention, allows for the determination of the thickness of a cover glass, under which the sample was placed, or the thickness of the bottom of a vessel in which the sample was placed, by means of the retaining focus unit. 
         [0028]    Such thickness determination can, for example, according to embodiments of the invention, be executed in such a way that the retaining focus unit illuminates the cover glass and/or the bottom of the vessel and thereby detects the reflections produced on the top side and the bottom side of the cover glass and/or the bottom of the vessel and therefrom derives the thickness to be determined. The detection of the reflections from top and bottom side are preferably detected under equal imaging conditions. For example, the two reflections can be mapped simultaneously on a focus measurement sensor of the retaining focus unit. 
         [0029]    For thickness determination, the retaining focus unit can furthermore, in embodiments of the invention, illuminate the cover glass and/or the bottom of the vessel and change the distance between the imaging optics and cover glass and/or the bottom of the vessel in such a way that the reflections produced on the top and bottom side of the cover glass and/or the bottom of the vessel are detected in temporal sequence and the thickness to be determined is derived while taking the change in distance into account. 
         [0030]    The microscopy method, according to embodiments of the invention, can further exhibit steps that are executed during the operation of the microscope (including its upgrades), according to the invention. 
         [0031]    It is understood that the aforementioned as well as the following characteristics still to be described are applicable not only in the described combinations but also in other combinations or unique characteristics without departing from the scope of the invention described herein. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0032]    In the following, the invention shall be further explained by way of examples through the attached drawings, which also disclose invention-essential characteristics. It is shown in: 
           [0033]      FIG. 1  is a schematic view of an embodiment of the microscope, according to the invention; 
           [0034]      FIG. 2  is a depiction for clarifying the determination of the magnification of the imaging optics  5 ; 
           [0035]      FIG. 3  is a depiction of the signals measured by means of the detector  8  during the determination in accordance with  FIG. 2 ; 
           [0036]      FIG. 4  is a depiction for clarifying the determination of the cover glass thickness; 
           [0037]      FIG. 5  is a depiction of the measurement results of the detector  8  for the determination of the cover glass thickness, according to  FIG. 4 ; 
           [0038]      FIG. 6  is a depiction for clarifying the cover glass thickness in accordance with a further embodiment; 
           [0039]      FIGS. 7   a  and  7   b  are depictions of the measuring signals of the sensor  8  for the determination of the cover glass thickness, according to  FIG. 6 ; 
           [0040]      FIG. 8  is a depiction for clarifying the determination of the focal position; and 
           [0041]      FIG. 9  is a depiction of the measuring signals of the sensor  8  for the determination of the focal position, according to  FIG. 8 . 
       
    
    
     DETAILED DESCRIPTION 
       [0042]    The embodiment in  FIG. 1  comprises the microscope  1 , according to the invention, which is preferably designed as an inverted microscope, a stage  2  which holds a sample  3 , an image sensor  4  as well as imaging optics  5 , which map the sample  3  onto the image sensor  4 . 
         [0043]    Moreover, the microscope  1  contains a motion unit  6 , which can adjust and readjust the distance between the imaging optics  5  and the stage  2  and therefore sample  3 , along the imaging direction of the imaging optics  5 . 
         [0044]    Between imaging optics  5  and the image sensor  4 , a partially transparent mirror  7  is positioned which allows the radiation for the mapping of sample  3  onto the image sensor  4  to pass almost unaltered and which deflects the focus measurement radiation (e.g., infrared radiation) onto the focus measurement sensor  8 . The focus measurement radiation is produced, e.g., with a radiation source (not shown), arranged in the area of the focus measurement sensors  8 , mapped via the mirror  7  and imaging optics  5  onto the sample  3 , reflected from the sample  3  and deflected via imaging optics  5  and the mirror  7 , as already mentioned, onto the focus measurement sensor  8 . The focus measurement sensor  8  emits a signal to the control unit  9  of the microscope  1 . In addition, the control unit  9  is connected with the motion unit  6 . 
         [0045]    With the motion unit  6 , the distance (in observational direction) between the imaging optics  5  and the stage  2  and therefore sample  3 , can be altered. This is indicated through lines L 2  and L 3 . Preferably, the motion unit  6  either moves only the imaging optics  5  or the stage  2 . This simplifies the mechanical configuration. 
         [0046]    The control unit  9  serves as control of the microscope and particularly for the execution of an image acquisition of the sample  3 . 
         [0047]    The image sensor  4  is connected to a control module not depicted herein. 
         [0048]    The control unit  9  is connected to an output/input unit  10 , which, e.g., can be designed as a touch-activated screen. 
         [0049]    Furthermore, the control unit  9  contains a retaining focus unit  11  with at least one hardware element  12  and one software module  13 . The retaining focus unit  11  can be set in a first position, whereby, for maintaining a predetermined focal position, it changes the distance between the stage  2  and the imaging optics  5  by means of the motion unit  6 , and in a second position, whereby it effects no change in distance. The retaining focus unit  11  is used preferably for microscopic examinations, which can temporally stretch over several hours or even several days, while set in the first position in order to ensure that the focal position is maintained permanently. 
         [0050]    As indicated in the schematic depiction in  FIG. 1 , the retaining focus unit  11  is, with regard to software as well as hardware, completely integrated in the control unit  9  of the microscope  1 . As a result, the retaining focus unit  11  can be operated via the output/input unit  10  and is part of the software environment of the control unit  9 , allowing for the retaining focus unit  11  to communicate directly with all system components of the control unit  9  and/or the microscope  1 . However, the system components can also communicate with the retaining focus unit  11 . For example, information from the retaining focus unit  11  can be prompted by the system components and used for the operation of the microscope  1 . 
         [0051]    Due to said complete integration of the retaining focus unit  11  in the control unit  9  of the microscope  1 , no further control panel for the retaining focus unit  11  is required. All functionalities of the retaining focus unit  11  can be operated via the output/input unit  10 . The selected operating conditions or also failure modes of the retaining focus unit  11  can, in turn, be displayed for the operator via the output/input unit  10 . The output/input unit  10  can therefore be used for the interactive control and/or adjustment of the retaining focus unit  11 . 
         [0052]    Therefore, in the microscope, according to the invention, the retaining focus unit  11  can be directly integrated in the control concept of the microscope  1  without making a further control panel necessary. 
         [0053]    The hardware element  12  of the retaining focus unit, which, e.g., can be a controller, can be connected to the internal bus system (not shown) of the microscope  1 . The retaining focus unit  11  can be integrated in the control unit  9  in such a way that the control unit  9  and the retaining focus unit  11  can be switched on and off separately from one another. 
         [0054]    If the control unit  9  is switched off and the retaining focus unit  11  is switched on, the retaining focus unit  11  will make a one-time attempt to contact the control unit  9  via the bus system. This is not possible due to the switched-off control unit  9 . 
         [0055]    If the control unit  9  is now switched on, it will query for existing components. In this case, the retaining focus unit  11  would log in. As a result, the respective outputs and displays on the output/input unit  10  are adjusted in order to be able to control the retaining focus unit  11  as well. 
         [0056]    If the control unit  9  is switched on but no contact is apparent with the retaining focus unit  11 , no output is produced on the output/input unit with regard to the retaining focus unit. Once the retaining focus unit  11  is switched on, the log in of the control unit  9  takes place via the bus system and as a result, the display on the output/input unit  10  is updated. 
         [0057]    Due to the integration of the retaining focus unit  11  in the control unit  9 , the retaining focus unit  11  can access all technically relevant data of the microscope  1 . Said technically relevant data include particularly the lens type with nosepiece position when the imaging optics  5  are designed as nosepiece with several lens systems, and the lens data, derived from the respective lens system, such as magnification, aperture, immersion, depth of field, applied wavelength range. 
         [0058]    Moreover, said technically relevant data can also include the applied illumination (e.g., reflected light, transmitted light, fluorescence), LSM activities (LSM=laser scanning microscope), laser exposure, z-position of the stage, the status of other components in the system as well as a utilized contrast method, if applicable (such as DIC=differential interference contrast, Ph=phase contrast, VAREL contrast, . . . ). 
         [0059]    In case of a connected incubator, the technically relevant data of the incubator (such as temperature) can, of course, also be taken into account. 
         [0060]    In addition, these components can be influenced directly by the retaining focus unit  11 . For example, the retaining focus unit  11 , if necessary, can effect a lens system change, activate a shutter and/or aperture, read and/or update (i.e., write) the ACR content (ACR=automatic component recognition) of lens systems, or even control the intensity of the illumination of the microscope. 
         [0061]    In order for the retaining focus unit  11  to maintain the predetermined focal position, knowledge about the imaging optics  5  is required, particularly the magnification of the imaging optics  5 . Under certain circumstances this might either not be known to the control unit  9  or the present entry may be incorrect. In such case, the retaining focus unit  11  would not be able to securely maintain the predetermined focal position. 
         [0062]    The retaining focus unit  11  provides the option of determining the magnification of the imaging optics  5 . 
         [0063]    Thereto, a glass plate with sufficient thickness (approx. 1 mm), preferably an object slide or a mirror, is placed as a sample on the stage  2 . Onto said sample  3 , it is focused in such a way that the imaging optics  5  is positioned in the right position, schematically shown in  FIG. 2 , relative to the sample  3 . On the focus measurement sensor  8  this leads to the right signal S 1  ( FIG. 3 ). 
         [0064]    Then the menu item for the determination of the magnification of the imaging optics  5  is activated via the output/input unit  10 . Consequently, the retaining focus unit  11  controls the motion unit  6  in such a way that the distance between the imaging optics  5  and the sample  3  is increased. The distance is increased until the signal on the focus measurement sensor  8  has moved from the right to the left side ( FIG. 3 ). The subsequently present signal S 2  is shown as dotted line in  FIG. 3 . Similarly, the present position of the imaging optics  5  is shown as dotted line in  FIG. 2 . 
         [0065]    From the change in distance Δz as well as the present signal shift AC on the image sensor  4 , the magnification v of the imaging optics  5  can be calculated with the following formula (1): 
         [0000]    
       
         
           
             v 
             = 
             
               
                 
                   k 
                    
                   
                       
                   
                    
                   
                     1 
                     · 
                     n 
                     · 
                     Δ 
                   
                    
                   
                       
                   
                    
                   C 
                    
                   
                       
                   
                    
                   μ 
                    
                   
                       
                   
                    
                   m 
                 
                 
                   Δ 
                    
                   
                       
                   
                    
                   z 
                 
               
             
           
         
       
     
         [0066]    This formula results for the concrete design of the microscope from  FIG. 1 , according to the invention, from the following formula (2): 
         [0000]    
       
         
           
             
               Δ 
                
               
                   
               
                
               z 
             
             = 
             
               
                 
                   sin 
                    
                   
                       
                   
                    
                   
                     α 
                     · 
                     n 
                     · 
                     k 
                   
                    
                   
                       
                   
                    
                   
                     2 
                     · 
                     Δ 
                   
                    
                   
                       
                   
                    
                   C 
                 
                 
                   2 
                   · 
                   
                     
                       ( 
                       
                         
                           f 
                           p 
                           ′ 
                         
                         / 
                         
                           f 
                           TL 
                         
                       
                       ) 
                     
                     2 
                   
                   · 
                   
                     v 
                     2 
                   
                 
               
               = 
               
                 
                   
                     
                       k 
                        
                       
                           
                       
                        
                       
                         1 
                         · 
                         n 
                       
                     
                     
                       v 
                       2 
                     
                   
                   · 
                   Δ 
                 
                  
                 
                     
                 
                  
                 C 
                  
                 
                     
                 
                  
                 μ 
                  
                 
                     
                 
                  
                 
                   m 
                   . 
                 
               
             
           
         
       
     
         [0067]    The formula (2) is based on the assumption that not depicted focusing optics in front of the focus measurement sensor  8  exhibit a test lens with the focal length f P  and a tube lens with the focal length f TL , that an angle α exists between the optical axis of the focusing optics and the vertical line of the focus measurement sensor  8 , that n is the refractive index of the immersion medium, and that the distance of adjacent pixels of the focus measurement sensor  8  equals k2. 
         [0068]    With the values of α=50°, f 1   P =16.559 mm, f TL =164.5 mm, and k2=6.7 μm, one arrives at the above formula (1) for magnification v, whereby k1=sin α·k2=253.26. The refractive index n for air is, e.g., 1, for water, e.g., 1.328, and for glycerol, e.g., 1.4477. 
         [0069]    The magnification v of the imaging optics  5  thus calculated is then utilized by the retaining focus unit  11  for the retaining focus control when, e.g., no magnification is available (for example, from the system data of the microscope) to the retaining focus unit. 
         [0070]    For example, it is also possible to have said determination of the magnification v of the imaging optics  5  effected automatically during the control of the retaining focus, in case certain threshold values of the control mode (speed, accuracy, etc.) are exceeded. 
         [0071]    Of course, it is also possible to display a menu item via the output/input unit  10  which allows the operator to have the described determination of the magnification of the imaging optics  5  carried out by the retaining focus unit  11 . The thus determined magnification can then, for the use of ACR lens systems, be stored in the respective ACR chip of the lens system. Of course, this can be carried out in dependence of a required input by the operator. 
         [0072]    The retaining focus unit  11  can be designed in such a way that it reads and utilizes all adjustment parameters required for the control when it is switched on by the operator. This can take place automatically and without further knowledge by the operator when, e.g., ACR lens systems are applied. In said lens systems, all required information is stored in the ACR chip assigned to the lens system. Hereby, the definition of the lens system with its adjustment parameters is automatically stored in the control unit  9  as soon as the operator activates the input. 
         [0073]    Alternatively, it is possible to read the ACR information anew at every lens system change and to store it in the control unit  9  in order to keep the lens entry as current as possible. 
         [0074]    Furthermore, it is possible to define the lens system manually via the output/input unit  10  or also via externally accessing software. Hereby, the assignment of the lens system can be simplified by entering the lens name or the order number. The required parameters for the retaining focus unit  11  are then concurrently entered automatically. 
         [0075]    It is also possible for the retaining focus unit  11  to store its obtained findings and/or parameters in the memory chip of the lens system insofar as an appropriate lens system is utilized. In addition or alternatively, the obtained findings and/or parameters can be stored in the system memory of the control unit  9 . In this case, said findings and/or parameters would also be available for externally accessing components. 
         [0076]    Frequently, the samples  3  are not placed directly on the stage  2 , i.e., a cover glass (for upright microscopy, not shown) is frequently placed between the sample  3  and the imaging optics  5 , or the bottom of a culture vessel and/or a Petri dish is placed between the sample  3  and the imaging optics, as, e.g., in inverted microscopy, indicated in  FIG. 1 . For a satisfactory image acquisition it is necessary to know exactly the thickness of the cover glass and/or the bottom thickness. In this case, the thereto designed lens systems of the imaging optics can be corrected accordingly with regard to the known thickness by means of a dial. 
         [0077]    In order to exactly determine said thickness, the two reflections, e.g., which are produced on both boundaries of the cover glass (hereinafter, the term cover glass shall apply to cover glass but also to the bottom of a vessel in which the sample is placed) to the surrounding medium  11  (as is indicated in  FIG. 4 ), can be evaluated. The two reflections R 1 , R 2  can be detected with the focus sensor  8 , whereby the reflection R 1  is the reflection on the top side of the cover glass DG, and the reflection R 2  is the reflection on the bottom side of the cover glass DG. The magnification of the imaging optics  5  must be selected in such a way that both reflections R 1 , R 2  are mapped on the focus measurement sensor  8 . 
         [0078]    From the distance ΔC between the two reflections R 1  and R 2 , available on the sensor  8 , the thickness d of the cover glass DG can be determined in accordance with the following formula: 
         [0000]    
       
         
           
             
               d 
               = 
               
                 Δ 
                  
                 
                     
                 
                  
                 
                   z 
                   · 
                   
                     
                       n 
                       DG 
                     
                     n 
                   
                 
               
             
             , 
           
         
       
     
         [0079]    whereby Δz results from the above formula (2) with the known value AC and the magnification factor v of the imaging optics  5 . n is once again the refractive index of the immersion medium and n DG  the refractive index of the cover glass. 
         [0080]    Alternatively, the magnification of the imaging optics  5  can be selected in such a way that only the reflection of the front or the rear boundary is detected by the focus measurement sensor  8 . Thereby, in a first position, preferably the reflection R 1  on the top side is initially determined ( FIGS. 6 and 7   a ). 
         [0081]    Then the distance between the cover glass DG and imaging optics  5  is changed in such a way that the reflection R 2  of the bottom side (dotted depiction in  FIGS. 6 and 7   b ) lies in the same position in the focus measurement sensor  8  as the first reflection R 1 . These steps are preferably executed automatically, e.g., upon request by the operator. 
         [0082]    From the thereto necessary distance change Δz, the cover glass thickness d can be calculated in accordance with the above formula 3. 
         [0083]    Focus tracking can be executed with the retaining focus unit  11  as follows. At first, the distance between the imaging optics  5  and the sample  3  is adjusted to a minimal value by the operator. As a result, the imaging optics are very close to the sample. Then the menu item focus tracking is selected via the output/input unit  10 . 
         [0084]    Then the retaining focus unit  11  controls the motion unit  6  in such a way that the distance between sample  3  and imaging optics  5  is increased. During said distance change, the retaining focus signals, determined by means of the focus measurement sensor  8 , are detected and stored preferably in defined path distances Δz KG . The path distances Δz KG  are preferably selected in such a way that the retaining focus signals of the focus measurement sensor  8  overlap in z-direction (observational direction). This is shown schematically in  FIG. 9  in such a way that the respective images of the focus measurement sensor  8  are depicted as overlapping. By means of the focus measuring signals M 1 -M 3 , the predetermined focal position is then determined which is utilized for the retaining focus control. For example, the maximum amplitude of the focus signal from the focus signals M 1 -M 3  as predetermined focal position can be inferred as the most likely focal plane and therefore predetermined focal position. 
         [0085]    If imaging optics are used whereby the lens system does not utilize an immersion medium and can therefore be called an air microscope lens system, at least two evaluable retaining focus signals, which correspond to the boundary between the upper glass bottom plane and the sample  3  (e.g., immersion liquid—usually water—of cells) as well as the boundary between the lower glass bottom plane and the air, can be detected while tracking the expected focus area. If the reflection of the boundary between the lower glass bottom plane and the air is used as reference area, which lies parallel to the focal plane, the focal plane must be approached offset by the bottom glass thickness. Thereto, the bottom glass thickness must be known, determined or entered. 
         [0086]    With water-immersed lens systems, two somewhat similar retaining focus signals can be detected. The first signal is very close to the focal plane and thereby recognized as the most likely focal plane. 
         [0087]    For immersion media, which exhibit a refractive index near the refractive index of the glass bottom, the reflection between glass bottom and immersion medium does not occur. In this case, the reflection between the sample (e.g., the immersion medium of the cells) and culture vessel material, which once again is very close to the focal plane, is detected with high probability. 
         [0088]    Preferably, evaluations by means of commonly applied retaining focus criteria are executed for exposures by means of the focus measurement sensor. Hereby, filter technologies and threshold value considerations can be applied which perform preselections in order to determine whether the detected signal is a retaining focus signal at all. Therefore, it is possible that, e.g., the retaining focus signal is detected in only one exposure M 2 . The other exposures M 1 , M 3  will signal that no focus can be expected therein. 
         [0089]    However, if the retaining focus unit  11  detects several areas (exposures M 1 -M 3 ) as possible focus, since retaining focus signals are also detected in these exposures, the areas and/or exposures can be evaluated in accordance with the aforementioned criteria, amplitude, noise performance, signal quality and/or the realistic evaluation of the plane to the starting point, etc.