Abstract:
A microscope objective including an front optical element, a plurality of optical elements spaced apart from the front element and from each other, as well as an adjusting unit. At least one of the optical elements can be displaced along the optical axis by the adjusting unit to adjust the focus of the objective. The focus of the objective is displaced relative to the front element along the optical axis and/or a temperature-induced imaging error of the objective is compensated for.

Description:
RELATED APPLICATION  
       [0001]     The current application claims the benefit of priority to German Patent Application No. 10 2005 034 441.0 filed on Jul. 22, 2005. Said application is incorporated by reference herein.  
       FIELD OF THE INVENTION  
       [0002]     The invention relates to a microscope objective used, for example, in fluorescence microscopy, in particular in 2-photon microscopy.  
       BACKGROUND OF THE INVENTION  
       [0003]     For experiments in fluorescence microscopy, e.g. during examination of living preparations, temperature control of the preparations is required. In order to avoid a temperature sink, the microscope objective is also heated in addition to the sample, so that the microscope objective is used, for example, in a temperature range of from 20 to 40° C. Since microscope objectives are often designed for a much narrower temperature range, spherical aberrations occur, for example, at temperatures outside said narrow temperature range, said aberrations causing the resolution to deteriorate.  
         [0004]     Further, in microscopy, in particular in laser scanning microscopes, optical sections are often performed at different depths of the sample. For this purpose, either the sample is moved along the optical axis of the microscope objective, or the entire microscope objective is moved. In doing so, the distance between the front lens and the sample surface is modified, which may lead to mechanical vibrations at high focusing speeds. If the microscope is operated with a liquid immersion medium, these vibrations may be disadvantageously transmitted to the sample.  
         [0005]     In view thereof, it is an object of the invention to provide a microscope objective by which the above-described difficulties can be overcome.  
       SUMMARY OF THE INVENTION  
       [0006]     According to the invention, the object is achieved by a microscope objective comprising an optical front element, a plurality of optical elements spaced apart from the front element and from each other, as well as an adjusting unit, at least one of said optical elements being displaceable along the optical axis by said adjusting unit such that the focus of the objective relative to the front element is displaced in the direction of the optical axis and/or a temperature-induced imaging error of the objective is compensated for.  
         [0007]     By modifying an individual distance or exactly one distance between two adjacent elements in the microscope objective, e.g. an undesired spherical aberration (aperture aberration) can be compensated for at least partially. Aperture aberration may occur, for example, when the microscope objective is used in an immersion microscope and the thickness of the immersion medium varies or the refractive index of the immersion medium is modified due to thermal changes. Replacement of the immersion medium with a different kind of immersion medium can also lead to such undesired aperture aberration.  
         [0008]     By modifying at least two distances, the aperture aberration, for example, can be very well compensated for. Further, the focus of the microscope objective relative to the front element of the microscope objective can be modified. In this case, if the microscope objective is used in a microscope, optical sections can thus be effected at different depths of the sample. This is advantageously achieved while maintaining the distance of the front lens to the sample surface and, thus, the working distance. The difficulties which usually result from modifying the working distance (for example, mechanical vibrations) can thus be completely avoided. The working distance is presently understood to be the distance of the front lens from the sample surface. Further, only few elements of the microscope objective, and not the entire microscope objective, need to be moved in order to achieve focusing as desired.  
         [0009]     Advantageously, the alteration of at least two distances in the microscope objective may also be used to compensate for temperature-induced spherical aberration. Thus, the range of applications for the microscope objective can be realized for much larger temperature ranges as compared with conventional objectives.  
         [0010]     Two or more distances of adjacent elements may be modified by the adjusting unit. In particular, the modification may be effected such that the distances can be respectively modified or adjusted independently of each other.  
         [0011]     The adjusting unit may comprise one or more adjusting elements. As adjusting elements, piezo elements may be used, for example.  
         [0012]     The change in distance is preferably effected in a controlled manner. In this case, a control unit is also provided, which accordingly controls the adjusting unit. The control unit may be, in particular, the control unit of the microscope in which the microscope objective is employed.  
         [0013]     Further, a temperature sensor may be provided, which measures (e.g. constantly) the temperature of the sample to be examined and/or of the microscope objective and transmits it to the control unit. The control unit then sets the distances as a function of the results of measurement.  
         [0014]     The optical front element of the microscope objective (i.e. that element whose distance from the sample is the shortest when using the microscope objective) may be, for example, a lens and may be arranged at the microscope objective, in particular such that it is not displaceable in the direction of the optical axis of the microscope objective.  
         [0015]     The microscope objective according to the invention may be used in a microscope, in particular a fluorescence microscope or a 2-photon microscope. In this case, a microscope is provided which can use the advantages of the microscope objective. For instance, the microscope may be an immersion microscope and/or a laser scanning microscope. Further, the microscope may comprise a control module which controls the adjusting unit of the microscope objective. In particular, the control module may be provided such that the microscopy method described hereinafter and its described further embodiments can be carried out with the microscope.  
         [0016]     Further, a microscopy method is provided, which uses a microscope objective comprising an optical front element and a plurality of optical elements spaced apart from the front element and from each other, wherein, in order to compensate for a temperature-induced imaging error and/or in order to modify the focus relative to the front element in the direction of the optical axis, at least one optical element is displaced along the optical axis. This displacement allows at least one distance between the elements spaced apart from each other to be selectively modified or adjusted, respectively, so that the desired compensation of the temperature-induced imaging error (for example, a temperature-induced spherical aberration) and/or the desired modification or adjustment, respectively, of the focus can be effected.  
         [0017]     In particular, the focus can be modified thereby in order to effect optical sections at different depths of the sample. Since the front element need not be moved for this purpose, the working distance remains constant during microscopy, thus avoiding the difficulties which result from modification of the working distance in a conventional approach, in particular when using immersion media.  
         [0018]     Therefore, it is advantageous to displace the at least one optical element such that the compensation of the imaging error and/or the modification of the focus are effected with the working distance remaining unmodified.  
         [0019]     In particular, the modification of the focus can be performed according to a given depth profile. This may be, for example, a periodic depth profile or any other given depth profile. The method then comprises tracking this depth profile (i.e. the focus is modified according to the depth profile), so that the optical sections can be obtained from the same depths in different samples (thus, microscopic photographs are respectively taken from the same depths in different samples).  
         [0020]     The method comprises displacing the at least one optical element such that two or more distances of the spaced apart optical elements are independently modified. In particular, an independent modification of exactly two distances allows to achieve an excellent displacement of the focus with at the same time negligible aberrations (caused by said displacement). 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0021]     The invention will be explained in more detail below, by way of example and with reference to the drawings, wherein:  
         [0022]      FIG. 1  is a schematic view of an embodiment of the microscope according to the invention, and  
         [0023]      FIG. 2  depicts a lens section of the microscope objective system of  FIG. 1 , in which the detection beam path of the objective is illustrated.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0024]     The microscope schematically shown in  FIG. 1  comprises a microscope objective system  1  and a source of illumination  2  which, in this case, can emit electromagnetic radiation at a wavelength of from 700-1100 nm.  
         [0025]     The microscope objective system  1  comprises an objective  3 , a color splitter  4 , detector optics  5 , as well as a surface detector  6  which has a circular detection area with a diameter of approximately 10 mm. The color splitter  4  is adapted to transmit the electromagnetic radiation from the source  2  and to reflect detectable radiation coming from the object or from the sample  7 , respectively, and lying in a wavelength range of from 400-700 nm.  
         [0026]     In operation, the radiation from the source  2  is transmitted by the color splitter  4  and focused on the sample  7  via the objective  3 . The detected radiation coming from the sample passes through the objective  3 , is reflected by the color splitter  4  to the detector optics  5  and is thereby directed onto the detector  6 .  
         [0027]     In the microscope objective system described herein, the objective is an immersion objective using water as the immersion liquid. The working distance D between the objective  3  and the sample  7  is 2.11 mm and the aperture is 0.8.  
         [0028]     The lens section of  FIG. 2  shows the beam path for the detection radiation which is directed onto the detector  6 . From the color splitter  4  up to the sample  7 , this beam path also corresponds to the illumination beam path for the light from the source  2 .  
         [0029]     The exact optical structure of the objective  3  and of the detector optics is indicated in the following Tables:  
                                                 TABLE 1                                   Surface—Surface   Distance [mm]   Material                                        F1-F2   2.0   Water           F2-F3   0.00           F3-F4   0.80   Suprasil           F4-F5   0.98   Gas (e.g. air)           F5-F6   17.37   N-LASF31           F6-F7   0.36   Gas (e.g. air)           F7-F8   2.50   Suprasil           F8-F9   0.99   Gas (e.g. air)            F9-F10   6.86   PSK3           F10-F11   0.05   Gas (e.g. air)           F11-F12   9.47   FK5           F12-F13   0.00   Cement           F13-F14   4.00   SF5           F14-F15   0.00   Cement           F15-F16   8.36   N-BaLF5           F16-F17   0.05   Gas (e.g. air)           F17-F18   17.46   N-LAK8           F18-F19   37.42   Gas (e.g. air)           F19-F20   23.00   Gas (e.g. air)           F20-F21   8.00   N-BK7           F21-F22   1.20   Gas (e.g. air)           F22-F23   8.00   N-BK7           F23-F24   2.00   Gas (e.g. air)           F24-F25   4.00   Filter           F25-F26   2.50   Gas (e.g. air)                      
 
         [0030]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
               
               
                   
                 Surface 
                 Radius of curvature [mm] 
                 Surface type 
               
               
                   
                   
               
             
             
               
                   
                 F1 
                 infinite 
                 planar surface 
               
               
                   
                 F2 
                 infinite 
                 planar surface 
               
               
                   
                 F3 
                 infinite 
                 planar surface 
               
               
                   
                 F4 
                 infinite 
                 planar surface 
               
               
                   
                 F5 
                 −14.236 
                 concave surface 
               
               
                   
                 F6 
                 −14.495 
                 convex surface 
               
               
                   
                 F7 
                 infinite 
                 planar surface 
               
               
                   
                 F8 
                 infinite 
                 planar surface 
               
               
                   
                 F9 
                 infinite 
                 planar surface 
               
               
                   
                 F10 
                 −26.120 
                 convex surface 
               
               
                   
                 F11 
                 42.398 
                 convex surface 
               
               
                   
                 F12 
                 −24.357 
                 convex surface 
               
               
                   
                 F13 
                 −24.357 
                 concave surface 
               
               
                   
                 F14 
                 18.042 
                 concave surface 
               
               
                   
                 F15 
                 18.042 
                 convex surface 
               
               
                   
                 F16 
                 −312.963 
                 convex surface 
               
               
                   
                 F17 
                 19.022 
                 convex surface 
               
               
                   
                 F18 
                 11.170 
                 concave surface 
               
               
                   
                 F19 
                 infinite 
                 planar surface 
               
               
                   
                 F20 
                 25.851 
                 convex surface 
               
               
                   
                 F21 
                 25.851 
                 convex surface 
               
               
                   
                 F22 
                 25.851 
                 convex surface 
               
               
                   
                 F23 
                 25.851 
                 convex surface 
               
               
                   
                 F24 
                 infinite 
                 planar surface 
               
               
                   
                 F25 
                 infinite 
                 planar surface 
               
               
                   
                 F26 
                 infinite 
                 planar surface 
               
               
                   
                   
               
             
          
         
       
     
         [0031]     The surfaces F 12 +F 13  as well as F 14 +F 15  are respectively cemented to each other. The element with the surfaces F  24  and F 25  is an emission filter.  
         [0032]     The surface F 7  is provided as a diffractive surface which can be described by the following phase polynomial P(r)  
         P   ⁡     (   r   )       ⁢     :     =       ∑     i   =   1     5     ⁢       a   i     ·     r     2   ·   i               
 
         [0033]     wherein a1=2.6647×10 −4 ; a2=3.985×10 −7 ; a3=1.3929×10 −9 ; a4=−3.1751×10 −13 ; a5=−3.7438×10 −17 , and r is the radial distance. The phase polynomial P(r) indicates the phase shift as a function of the radial distance r, and the grating frequency of the diffractive element can be calculated on the basis of the derivation of the phase polynomial according to the radial distance r.  
         [0034]      FIG. 2  further schematically shows two adjusting elements  11  and  12 , which together form an adjusting unit. As indicated by the double arrow P 1 , the adjusting element  11  allows the element with the surfaces F 7  and F 8  to be moved along the optical axis OA of the objective  3 . Likewise, the group of optical elements with the surfaces F 9 -F 18  can be moved together in the direction of the optical axis of the objective  3  by means of the adjusting element  12  as indicated by the double arrow P 2 . This makes it possible to independently adjust the distance between the surfaces F 6  and F 7  and the distance between the surfaces F 8  and F 9  by means of the adjusting elements  11  and  12 . This may be utilized in order to modify the focus along the optical axis without modifying the working distance D, so that optical sections can be effected at different depths of the sample. Since the working distance D need not be changed for this purpose, this does not lead to otherwise disadvantageously appearing vibrations and transmissions of force onto the sample via the immersion liquid, which would occur during conventional focusing due to the movement of a specimen stage and/or of the objective and, thus, due to the modification of the working distance.  
         [0035]     The necessary modification of the distances between the surfaces F 6  and F 7  as well as between the surfaces F 8  and F 9  for a defocusing range of +/−0.1 mm is indicated in the following Table 3.  
                                     TABLE 3                       Defocusing   Modification of Distance   Modification of Distance F8-F9       [mm]   F6-F7 [mm]   [mm]                                −0.1   −0.16   0.85       −0.08   −0.12   0.68       −0.06   −0.09   0.51       −0.04   −0.06   0.34       −0.02   −0.03   0.17       0.0   0.0   0.0       0.02   0.03   −0.17       0.04   0.06   −0.34       0.06   0.09   −0.51       0.08   0.12   −0.68       0.10   0.15   −0.85                  
 
         [0036]     It is further possible to achieve temperature compensation of imaging errors within a temperature range of, for example, from 20 to 40° C. by modifying the distances F 6 -F 7  as well as F 8 -F 9 . If the objective  3  is designed for a temperature of 20° C., it is required, for an operating temperature of 30° C., to modify the distance F 6 -F 7  by −0.0324 mm and the distance F 8 -F 9  by 0.0109 mm. For a temperature of 40° C., the modification of the distance F 6 -F 7  is −0.0658 mm and the modification of the distance F 8 -F 9  is 0.0231 mm.  
         [0037]     As adjusting elements  11  and  12 , piezo-adjusting elements or other adjusting elements having the desired precision for displacement of the elements can be used.  
         [0038]     In particular, a temperature sensor (not shown) can also be provided, which constantly measures the temperature of the objective  3  and transmits it to a control unit (not shown). As a function of the measured temperature, the control unit then controls the adjusting elements  11  and  12 . Of course, such control unit may also be used to modify the focus in the above-described manner.  
         [0039]     In the microscope shown in  FIG. 1 , e.g. a deflecting unit may also be arranged between the objective  3  and the color splitter  4 , so that the microscope is then provided as a laser scanning microscope. The detector optics  5  may also be omitted.