Abstract:
A laser-micro-dissection method and a device for laser micro-dissection involves cutting a dissectate from a biological sample, which is applied to a planar carrier, by means of laser pulses along a closed cutting line. The parameters, which determine the laser pulses and the cut lines, are synchronous in relation to the laser pulses and are continually modified along the closed cut line. All elements which are arranged in the optical axis and which determine the parameters of the laser pulse and the cut lines, are controlled by a central calculation unit.

Description:
BACKGROUND 
     The invention relates to a laser microdissection method with the aid of which a dissectate is cut out along a closed cutting line from a biological specimen, which is mounted on a planar carrier, by means of laser pulses of a laser. 
     Furthermore, the invention relates to a device for laser microdissection that comprises a microscope having at least one objective defining an optical axis. Furthermore, a pulsed laser is provided that emits a laser beam that is directed along the optical axis onto a specimen via the objective and describes a closed cutting line. 
     In the field of biology and medicine, microdissection denotes a method with which a small piece, a so-called dissectate, is cut out of a generally flat specimen (for example cells, cell cultures or a tissue section) with the aid of a focused laser beam. The biological specimen is mounted for laser cutting on a planar carrier, for example a glass specimen slide or a polymer film. The dissectate is available after the cut for further biological or medical (for example histological) examinations. 
     Such a method for a laser microdissection is described in the article entitled “Cell surgery by laser microdissection: a preparative method” by G. Isenberg, W. Bilser, W. Meier-Ruge, E. Remy, Journal of Microscopy, vol. 107, May 1976, pages 19-24. A biological specimen is mounted there on the underside of a specimen slide. What is meant by biological specimen is cell cultures that have been attracted on a specimen slide. In order to prevent a permanent adhesion of these cells on the substrate, use is made of silicone-coated specimen slides that effect a reduction in the adhesion between specimen and specimen slide. The specimen slide lies in an erect microscope into which a pulsed He—Ne laser is coupled. The laser beam is focused onto the biological specimen. A specimen field of interest, the dissectate, is cut out along a closed cutting line by juxtaposing cut holes produced by the laser pulses with the aid of the focused laser beam. The cutting is based in this case on the known principle of laser ablation, that is to say the individual laser pulses produce on the cutting line a plasma that “vaporizes” the specimen material. In this case, the last laser pulse separates the dissectate from the surrounding biological specimen and in so doing it also effects the required loosening of the dissectate from the specimen slide. The dissectate then falls down under the action of gravity, and is captured in a collecting vessel and fed to further examinations. 
     DE 100 43 506 C1 describes a further development of this method. In this case, the specimens to be examined and from which specimen fields of interest are to be cut out are prepared on very thin plastic films. The thickness of these plastic films is of the order of magnitude of 1-2 μm. PET films and PEN films come into question as material. The specimen is loaded into a microscope into which a pulsed laser is coupled. A method for laser microdissection is described in which the cutting line is not completely closed toward the end of the cut, but a narrow and at the same time stable web remains at the end. This prevents the film with the specimen field of interest from being swung out and twisted outside the focal plane. Before the web is severed, the aperture of the laser beam is enlarged by means of a diaphragm without varying the observation aperture of the microscope. The cutting width of the laser beam is enlarged by the enlarged laser aperture. At the same time, the position of the focus of the laser beam is kept without variation at the same position relative to the specimen. The residual web is then severed with the expanded laser aperture with the aid of a last, focused, cutting laser pulse. At the termination of the cut, the specimen falls down under the action of gravity and is collected in a collecting vessel. However, it has emerged overall that it is complicated in terms of equipment and time-consuming to stop the cutting line before the last laser pulse and to switch over the diaphragm for the laser aperture before the cutting line is terminated with the last laser pulse. Again, it proves not to be quite so simple for the user to fix a suitable residual web and to assign a fitting laser aperture, and so the dissectates are sometimes not entirely freely prepared and the cut has to be repeated. 
     Moreover, it has been observed in the case of both methods that when the equipment setting (optics, laser parameters, focal position etc.) is not varied, dissectates sequentially cut out drift away laterally to a different extent when they fall into the collecting apparatus. This collecting apparatus can be, for example, a specimen tube, usually referred to in the market as a PCR tube. The consequence is then that the dissectates adhere laterally to the inner wall of the PCR tube instead of falling to the bottom of the tube. It is then difficult for them to be inspected, and this constitutes for the user, for example a pathologist, a substantial working step before the further processing of the cutout dissectates. 
     Consequently, German patent application DE 103 46 458 proposes a method for laser microdissection of a specimen field of interest of a specimen in which the laser pulses of a pulsed laser beam are likewise focused on the specimen, and in the case of which the mass ablated at the last laser pulse completing the cut is adapted to the cutting width of the last cutting laser pulse and optimized so as to maximize the energy transferred from the plasma on to the dissectate. 
     However, the stopping of the cutting line before the last laser pulse is felt to be time-consuming by the user here too. 
     U.S. Pat. No. 6,773,903 likewise discloses a method for microdissection in which selected fields of a biological specimen are cut out. The specimen mounted on the specimen slide lies on a stage movable in the x-y coordinate plane. A laser beam is coupled into the microscope and the x-y stage is appropriately moved such that this laser beam describes an appropriately closed cutting line about the specimen field of interest. Consequently, the biological material of interest is separated from the biological specimen. The control of the x-y stage is, however, mechanically complex and not so accurate as if the laser beam were controlled appropriately in the x-y plane in order to separate the biological material from the remainder of the specimen. 
     It is therefore an object of the invention to specify a method for laser microdissection that permits the dissectate to be cut out in a more comfortable and speedier fashion accompanied by further improved cutting results even in the case of difficult specimen preparation. 
     This object is achieved by a laser microdissection method described herein. 
     A further object of the invention is to specify a device for laser microdissection with which the user can obtain the desired dissectates precisely, quickly and reliably. In this case, obtaining the dissectates is independent of the respective specimen preparation. 
     This object is achieved by a device for laser microdissection described herein. 
     SUMMARY 
     In the case of the inventive laser microdissection method, a dissectate is cut out from a biological specimen by means of laser pulses of a laser beam. In this case, the laser beam is guided along a closed cutting line. The specimen itself is mounted on a planar carrier. While the dissectate is being cut out, parameters that determine the laser pulses and the cutting line are continuously varied along the closed cutting line. 
     The continuous variation of the parameters along the closed cutting line is determined by image processing. The variables for the continuous variation of the parameters along the closed cutting line are obtained from the image processing. These variables are, for example, the specimen thickness, the texture of the specimen, the distribution of the staining inside the specimen, etc. 
     It is likewise conceivable that the parameters that determine the laser pulses and the closed cutting line are continuously varied only before a closure of the closed cutting line. The parameters remain constant during the rest of the cutting of the specimen. 
     A slider can be used on a user interface to vary the parameters that determine the continuous variation before the closure of the closed cutting line. 
     The parameters are determined by means of a central processor, the central processor supplying corresponding control signals to the individual elements of an optical system. 
     The laser pulses traverse the optical system before they strike the biological specimen, the parameters of the laser pulses being varied with regard to an aperture, an attenuation, a density of the individual laser points on the cutting line and a focal position of the laser pulses. 
     The aperture and the attenuator are varied simultaneously. The variation of the aperture and of the attenuator is performed synchronously with the laser pulses in order thereby to obtain the highest cutting speed. The variation of the aperture can be carried out by means of a pinhole diaphragm or an iris diaphragm. 
     The density of the individual laser points inside a cutting line can be adapted with regard to the respective laser power and the local properties of the specimen. 
     The inventive device for laser microdissection comprises a microscope having at least one objective defining an optical axis. Likewise provided is a pulsed laser that emits a laser beam that is directed along the optical axis onto a specimen via the objective. The laser beam describes a closed cutting line on the specimen in order thereby to separate a selected field of the specimen from the rest of the surrounding biological material. All the elements arranged on the optical axis, which determine the parameters of the laser pulses and the cutting line, are connected to a central processor. 
     By means of image processing the central processor attains a continuous variation of the parameters along the closed cutting line via a correlated adjustment of the individual elements. 
     In this case, the central processor can likewise be used such that the central processor continuously varies the parameters that determine the laser pulses and the cutting line only before a closure of the closed cutting line. 
     The elements that determine the parameters of the laser pulses and the cutting line are an X/Y displacement unit, an aperture unit, an attenuator unit, a focusing unit, a UV laser and a deflecting unit. The central processor in this case supplies appropriate adjusting signals to the respective elements. 
     Likewise connected to the device for laser microdissection is a monitor on which a user interface is displayed to the user. The user interface constitutes a slider with which the continuous variation of the parameters can be determined before the closure of the closed cutting line. The central processor in this case varies the aperture and the attenuator simultaneously. 
     Further advantageous refinements of the invention can be gathered in this case from the subclaims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention is described more accurately below with reference to the schematics, in which: 
         FIG. 1  shows a device for laser cutting with the aid of a stationary laser beam; 
         FIG. 2  shows a device for laser cutting with the aid of a movable laser beam; 
         FIG. 3  shows the parameter composition in accordance with the prior art in the case of which the parameters remain constant up to the end of the cutting line; 
         FIG. 4  shows a parameter composition in accordance with the prior art in the case of which the parameters are varied suddenly before the end of the complete cutting out of a specimen; 
         FIG. 5  shows a continuous variation of the parameters along a closed cutting line; 
         FIG. 6  shows a continuous variation of the parameters before the end of the cutting line; 
         FIG. 7  shows a schematic of a cutting line over the course of which the parameters are varied continuously in the course of the entire cutting line; 
         FIG. 8  shows a schematic of the cutting line in the case of which the parameters are not varied before the end of a cutting line; 
         FIG. 9  shows the overall efficiency of the variation of the aperture A and of the attenuator K; 
         FIG. 10  shows an optimized overall efficiency from the variation of the aperture A and of the attenuator K; 
         FIG. 11  shows cutting lines in the case of which the aperture diaphragm and the attenuator have been adjusted in common; 
         FIG. 12  shows, by way of example, the restrictions of the possible combinations of an attenuator with a dynamic range of 40:1 and an aperture diaphragm of the same dynamic range; 
         FIG. 13  shows, by way of example, control curves for attenuator and aperture that are in no way restricted just to profiles with monotonic variation of the curvature; 
         FIG. 14  shows a typical calibration curve as a function of the angular position of the attenuator; 
         FIG. 15  shows a linearization of the characteristic of the attenuator from a prescribed control value; 
         FIG. 16  shows, by way of example, a schematic of a program loop for generating a cutting line; and 
         FIG. 17  shows a display of a section from a user interface via which the continuous variation of the parameters before the closure of the closed cutting line is set by means of a slider. 
     
    
    
     DETAILED DESCRIPTION 
     Identical elements are denoted in the figures by the same reference numerals. 
       FIG. 1  illustrates a device for microdissection that operates with the aid of a stationary laser beam and a specimen  4  moved relative thereto. The device comprises a microscope  1  having an x-y stage  2  that can be moved by motor. The x-y stage  2  serves to receive a specimen holder  3  on which a specimen  4  to be examined and/or cut is mounted. Also provided is an illumination system  5  with which the specimen  4  is possible for visual observation by the user via an eyepiece  12 . In order to cut the specimen  4 , a laser beam  22  is provided that is coupled into the optical axis  10  of the microscope. The laser beam  31  produced by the laser  22  is focused onto the specimen  4  for the purposes of cutting. The x-y stage  2  is connected to a control unit  15  that moves the x-y stage  2  in such a way that the desired cutting line is generated. The appropriate specimen part is then cut out of the specimen by means of the desired cutting line by means of the relative movement between the laser beam  31  and the specimen  4 . The microscope illustrated in  FIG. 1  is a transmitted-light microscope in the case of which the illumination system  5  is arranged on a microscope stand  8  below the x-y stage  2  and the specimen  4 . The microscope  1  comprises at least one objective  9  that is arranged above the x-y stage  2  and the specimen  4 . The objective defines an optical axis  10  that is aligned with the optical axis of the illumination system  5 . In this described arrangement, the specimen  4  is viewed with the aid of a transmitted-light illumination. The laser cutting could also likewise be executed with the aid of an inverse microscope in the case of which the illumination system  5  is arranged above the x-y stage  2 , and that at least one objective is arranged below the x-y stage  2 . The light emanating from the illumination system  5  is directed from below by a condenser  11  onto the specimen holder  3 , arranged on the x-y stage  2 , with the specimen  4 . The light penetrating the specimen  4  passes to the objective  9  of the microscope  1 . Inside the microscope, the light is fed to the at least one eyepiece  12  via lenses (not illustrated) and mirrors. Likewise connected to the microscope  1  is a camera  17  that records an image section of the specimen  4  as a function of the magnification of the objective. The image data recorded by the camera are passed on to a processor  16  that, for, its part, is connected to a monitor  18  on which an image of the recorded specimen field can be displayed to the user. A control unit  15  is likewise interposed between the camera and the processor. The laser beam  31  emanating from the laser  22  is coupled into the beam path  10  of the microscope via a beam splitter  13 , such as, for example, a dichromatic splitter. Before the laser beam  31  is coupled into the beam path of the microscope  1 , it traverses an optical system in which a number of elements  14 ,  16  and  19  are provided. The first element in the optical system is an aperture unit  14  that is connected to a control unit  15 , that for its part is connected in turn to the processor  16 . The aperture unit  14  can comprise an iris diaphragm or a selection from a number of different pinhole diaphragms. The second element  19  in the optical system  30  is a focusing unit  19  that is for its part likewise connected to a control unit  15  that is likewise connected to the processor  16 . The focusing unit  19  serves chiefly for balancing the different focal positions in the ultraviolet of the objectives  9  of the microscope  1 , that are chiefly corrected in the visible spectral region. Alternatively the focusing unit  19  can also be used to select a specific focal position or a continuous variation of the focal position of the laser focus during the cutting operation. The third element  16  in the optical system  30  is an attenuator unit  16 . 
     The attenuator unit  16  is likewise connected to a separate control unit  15  that, in turn, is connected to the processor. The laser beam  31  is produced by a UV laser  22  that is introduced into the optical system  30 . The UV laser  22  is likewise connected to the processor  16 . 
     The attenuator unit  16  can be varied in angular position, and the attenuation is based on the principle of interference. The aperture unit  14 , the focusing unit  19  and the attenuator unit  16  can all be varied in their position and/or size by control signals from the individual control units  15 . The variation is performed in this case by a motor. The aperture unit  14 , the focusing unit  19  or the attenuator unit  16  is connected to the individual control unit  15  that, on its part, is connected to the processor  16  via an individual feedback line  15   a . The aperture unit  14 , the focusing unit  19  and the attenuator unit  16  can in this case be adjusted independently of one another. The independent adjustment of the aperture unit  14  and the attenuator unit  16  yields in combination a maximum dynamic range (variation width of the laser power in the specimen), and the variables such as depth of field, resolution and power density can be influenced in a targeted fashion independently thereof. The specimen  4  is cut with the aid of individual laser pulses using the laser beam  31  coupled into the optical system  30 , which is reflected into the beam path  10  of the microscope  1  via a beam splitter. However, an optimum cutting result for the purpose of the invention requires the synchronization of the laser pulses with the variations of all of the aperture unit  14 , the attenuator unit  16  and the focusing unit  19 , there also being a need to take account of the movements of the x-y stage  2 . 
       FIG. 2  shows a device for laser microdissection in the case of which the x-y stage is stationary and the laser beam is deflected in the appropriate way by a deflecting unit  40 , that is likewise arranged in the optical system  30 , in order to cut out from the specimen  4  a dissectate of any desired shape. In this arrangement the x-y stage  2  cannot be moved during the cutting operation. Proceeding in the direction of the laser beam emanating from the laser  22 , the arrangement in the optical system  30  is: firstly the attenuator unit  16 , then the focusing unit  19 , then the aperture unit  14  and, finally, the deflecting unit  40 . The deflecting unit  40  is connected to an individual control unit  15  that, for its part, is connected to the processor  16  via a feedback line  15   a . The deflecting unit  40  consists of a pair of wedge plates that can be displaced in a suitable way by the control unit  15  in conjunction with the processor  16  so that the laser beam describes on the specimen the desired shape that the cutout dissectate is finally intended to have. As already mentioned in the description relating to  FIG. 1 , all the elements of the optical system  30  are each connected to individual control units  15  that are connected to the processor  16  via a feedback line  15   a.    
       FIG. 3  shows the set of parameters as used in the case of a cutting method for dissectates in accordance with the prior art. The cutting length in percent is plotted on the abscissa. In this case, 0 percent signifies the beginning of the cut and 100 percent the end of the cut. The individual parameter values are plotted on the ordinate  33  in arbitrary units as a function of the cutting length. All the parameters such as, for example, focus  34 , point spacing  35 , attenuator  36  and aperture  37  are constant over the entire cutting length. The total power  38  that results from the interaction of attenuator  36  and aperture  37  is therefore likewise constant over the entire cutting length. 
       FIG. 4  likewise describes the combination of parameters during a cutting process of a dissectate in accordance with the prior art. Cutting length is likewise plotted in percent on the abscissa  41 , and the value of the parameters is illustrated in arbitrary units on the ordinate  42 . The focus  43 , the point spacing  44 , the attenuator  45  and the aperture  46  are constant over virtually the entire cutting length. As already described in  FIG. 3 , the parameters of the attenuator  45  and the parameters of the aperture  46  yield a total power  47  that is therefore likewise constant up to shortly before the end of the cutting line. Shortly before the end of the cutting length or cutting line, the system inserts a short pause in which the parameter of the aperture  46  is varied. The aperture is therefore enlarged before the end of the cutting line. Consequently, there is thus also a change in the total power that therefore also becomes larger owing to the large aperture. The other parameters such as, for example, attenuator  45  and focus  43  remain constant in this case. 
       FIG. 5  shows a set of parameters that vary continuously over the entire cutting length. The cutting length is plotted in percent on the abscissa  50 , and the continuously varying values of the individual parameters are plotted on the ordinate  51 . As may be seen from  FIG. 5 , the parameters of the focus  52 , the point spacing  53 , the attenuator  54 , the aperture  55  and therefore also the total power  56  vary over the entire cutting length. A variation of the focal position is required in order to balance an oblique position of the specimen, or in order to adapt the focus to different thicknesses of the specimen. Likewise, with the aid of the changing focus it is possible to select a specific z-position or a continuous variation of the z-position of the laser focus during the cutting operation or the production of a cutting line. The cutting line is produced by a juxtaposition of individual laser pulses. It is important in this case that the points touch one another in order thereby to produce a cutting line that separates the dissectate from the rest of the specimen  4 . As illustrated in  FIG. 5 , it is likewise possible to this end to vary the point spacing of the individual laser pulses inside the cutting line. A variation in point spacing signifies the diameter of the individual laser pulse can change during the production of the cutting line. The interaction of the aperture  55  and the attenuator  54  likewise results in a continuously changing total power  56  that is input onto the specimen  4  by the laser pulse. In the case described in  FIG. 5 , the variables of total power  56 , aperture  55 , attenuator  54 , point spacing  53 , as well as the focal position  52  are varied continuously and synchronously with the laser pulses in order to achieve a detachment that is as reliable as possible in conjunction with an optimized cutting speed. Estimated values for these parameters can be determined, for example, by evaluating the optical density in the entire spectral region or in specific spectral regions or color channels, and by “calibrating” the method to the specimen material, that is to say by cutting trials in a part of the specimen not otherwise used. In this case, there is the additional freedom of achieving the same total laser powers from different settings of attenuator and aperture diaphragm, and thus, depending on the section of the cutting line, of selectively, for example, optimizing at (a) or (b) either the depth of field (small aperture) or the power density at the focus (large aperture). A possible curvature and a general inclination of the preparation relative to the optical axis can also be determined (c) via evaluation of the contrast of the microscope image at various focal positions, and can be varied within the scope of the proposed invention simultaneously with the laser pulses and thus without reducing the cutting speed. Estimated values for the cutting properties of the specimen material along the prescribed cutting curves l and l i  are then optionally determined for example by evaluating the optical density d in the entire spectral range or in specific spectral ranges or color channels, or else with the aid of other methods. A profile of the cutting parameters such as total laser power P (see below), aperture A, attenuator K, point density D and, if appropriate, also focal position z along l is determined and stored for the subsequent cutting process on the basis of the optical density d thus estimated, together with user data relating to the type of the specimen material or carrier material x. A few prescribed single parameter functions (P, D, z)=f x (d) or else (A, K, D, z)=f x (d) are also conceivable here for the purpose of simplifying the operation. Alternatively, it is also possible to determine the best cutting parameters by “calibrating” the method to the specimen material, that is to say by means of cutting trials in a part of the specimen  4  not otherwise used. 
     A possible curvature and a general inclination of the preparation or specimen  4  to the optical axis  10  can also be determined by evaluating the contrast of the microscope image at various focal positions and be varied in the scope of the proposed invention in a fashion simultaneous to the laser pulses and thus without reducing the cutting speed. 
       FIG. 6  describes the situation in which some parameters are changed continuously shortly before the end of the cutting line. The cutting length is plotted in percent on the abscissa  60 , and the values of the individual parameters are illustrated in arbitrary units on the ordinate  61 . The changing parameters are the focus  62 , the point spacing  63 , the attenuator  64 , the aperture  65  and the total power  66 . In this case, the focus  62  can remain constant over the entire cutting line or change continuously over the entire cutting line. Starting from approximately 60 percent of the terminated cutting line, there is a continuous increase in the values of remaining parameters such as point spacing  63 , attenuator  64 , aperture  65  and, consequently, the total power  66 . The previously mentioned values of the parameters are constant up to 60 percent of the cutting line. As a result, a reliable separation of the dissectate from the remainder of the specimen material or the carrier is achieved by a continuous increase in the total power acting on the specimen  4  before the end of the cutting line. An explicit determination of the cutting parameters along the cutting line is dispensed with in the case of the parameter setting shown in  FIG. 6 . The aim is as reliable as possible detachment of the dissectate in conjunction with the high “overall” cutting speed. No part of the cutting curve is omitted, but laser power (or aperture and attenuator) and point spacing are continuously increased in the end region of the cutting curve in order to minimize premature lowering or, as caused by stresses, setting up of the dissectate from the focal position. These variations are described by classes x of single-parameter functions that take the profile of laser power and point spacing along the cutting line. The selection of x can be performed on the basis of typical (known or previously determined) material properties, or else by a type of “calibration”. Alternatively, or else in addition, it has proved to be helpful to the user to influence the selection of the correct function class x via an additional setting variable that describes how critical the respective material is with reference to the interfering effects such as setting up, lowering, tilting, or sliding up and bonding, and that then, for example, prescribes the variations of the total laser power (or of the aperture and the attenuator) and of the associated point density toward the end of the cutting line. The user can thus distinguish in as simple a way as possible between uncritical specimen materials on the one hand, and specimen materials that are difficult to separate on the other hand, and adapt the cutting process correspondingly. 
     In the simplified case of  FIG. 6 , no optical density d is determined along the cutting line l and used, but an optimized parameter profile (P, D)=g x (l) or else (A, K, D, z)=g x (l) is determined, g x  again being classes of single-parameter functions that describe the profile of the parameters along the cutting line l, particularly toward the end, in order, as already discussed further above, to achieve both cuts as thin as possible over as large as possible a part of the cutting line l, and as reliable as possible a detachment in the critical end region of l. Selection of x can be performed on the basis of typical (known or previously determined) material properties, or again by means of a type of calibration, as already discussed in the description relating to  FIG. 5 . Alternatively, or else in addition, it has proved to be helpful to the user to influence the selection of the correct function g x  via an additional setting variable that describes how critical the respective material is with reference to the abovementioned interfering effects such as tilting, sliding up, bonding or bending up, and that then prescribes, for example, the variations of the total laser power P (or A and K) and the associated point density D toward the end of the cutting line. The user can thus distinguish in as simple a way as possible between uncritical specimen materials on the one hand, and specimen materials that are difficult to separate on the other hand, and optimize the cutting process correspondingly. 
     It is also optionally possible in the cutting method described in  FIG. 6  to use contrast evaluation of the microscope image in various focal positions to determine a possible curvature and a general inclination of the preparation relative to the optical axis, and simultaneously to correct the laser pulses without reducing the cutting speed. 
       FIG. 7  shows a cutting line  70  inside which a number of regions are provided in which the combination of the parameters of the laser pulses on the specimen  4  change. In the present embodiment, the cutting line  70  is subdivided into four regions  71 ,  72 ,  73  and  74 . Thus, for example, cutting is conducted with constant parameters in the region  71  and in the region  73 . In region  72  and in region  74 , the parameters for producing the cutting line vary continuously. Either the user employs a mouse (not illustrated) to mark the desired cutting line l directly in the image of the specimen  4  on the monitor  18  or, if appropriate, he roughly prescribes just a search region, and an image detector determines in a fully automatic fashion one or more/all cutting lines l i  in the prescribed region of the specimen  4 . 
       FIG. 8  shows a further embodiment for producing a cutting line  80 . The cutting line has a start that is denoted by  81  in  FIG. 8 . The start of the cutting line  81  coincides with the end of the cutting line  83 . Proceeding from the start  81  of the cutting line, cutting is performed with constant parameters up to a position  82  on the cutting line. The constant parameters are applied in the cutting line between the start  81  and a position  82  in the cutting line. The parameters are then continuously varied or raised between the position  82  and the start or end  81  respectively of the cutting line. Thus, after approximately 60 percent of a completed cutting line, at the earliest, a start is made on continuously varying the cutting parameters up to the end of the cutting line. During the cutting process, the mean width of the cutting line is a few μm. 
       FIG. 9  shows the overall efficiency in the case of simultaneous variation of the aperture and the attenuator. The control value is plotted in arbitrary units on the abscissa  90 . The intensity is illustrated in logarithmic units on ordinate  91 . Simultaneous variation of aperture and attenuator is required in order to achieve as large a dynamic range as possible. The combined driving of the attenuator and aperture raises the resolution in the case of the power setting. The diaphragm steps are usually only coarsely logarithmically graduated for the aperture. One diaphragm step corresponds to a variation in intensity of &gt;30 percent. The attenuator can, in contrast, be set with a resolution of 7 percent and better (smaller microsteps are possible). A high dynamics (total dynamics of approximately 1:2000) can be achieved by the combined simultaneous driving of aperture and attenuator in conjunction with a resolution of better than 7 percent (100 steps at 0.928). The variation  92  of the attenuator is illustrated with discrete steps in  FIG. 9 . The variation  93  of the aperture is likewise illustrated in discrete steps in  FIG. 9 , the steps being larger in the case of the aperture setting than in the case of the setting of the attenuator, as already mentioned above. The overall efficiency of the intensity resulting from the combined driving of the aperture and the attenuator is illustrated in the curve  94 . It follows that the curve  94  shows periodic jumps in the rise of intensity in the overall efficiency. During a cutting operation, both the aperture A and attenuator K of the laser beam  31  are varied arbitrarily. However, not all combinations of A and K are in practice independent or sensible for a specific application. In principle, there are a number of combinations of A and K that all lead to the same total power input P into the specimen, and by analogy with photography, many combinations of diaphragm B and exposure time t lead to the same exposure of the film. If A denotes the logarithm of the aperture diameter, and K the logarithm of the transmission of the attenuator, it holds in simplified fashion that P=A+K (with P also in logarithmic units), it thereby being clear that (infinitely) many values of A and K can lead to the same sum P. However, it also holds true here by analogy with photography that the end result can by all means be different even when the mean power import P (the mean exposure in the photograph) is the same. In photography, these properties are taken into account by so-called “program automatics”, that is to say the values of B and t are varied over the useful range of the exposure in a specific scheme and, depending on application, the respective scheme is changed or adapted, for example, concerning whether maximum depth of field or whether minimum movement of field is desired. The idea of a “program automatic”, that is to say a scheme according to which A and K are varied together, can now be used together with the method described here and the associated device for an improvement of power control in laser microdissection. 
     The dynamics ranges that can be attained alone with the aid of aperture stop and attenuator are restricted in practice to values of approximately 70:1 or 30:1 in a particular case. However, an optimum cutting quality typically requires the variation of the overall efficiency over a relatively large range. Very large power changes can also be implemented without loss of speed through simultaneously varying the aperture stop and attenuator synchronously with the laser pulses, because the adjustment path of the individual components is less than when the change would have to be implemented solely with the aperture stop and the attenuator alone, and a dynamics range of=2000:1 overall is achieved after all. 
     In the simplest case (see  FIG. 9 ), aperture and attenuator are varied monotonically over the prescribed range of a control value in order to implement this large dynamic scope. However, in some circumstances this gives rise to unequally large power steps (as already mentioned above). The finer graduation of the attenuator in the example therefore cannot be utilized against the coarsely graduated aperture. 
       FIG. 10  likewise shows a combination of the overall efficiency of attenuator and aperture. Here, the control value is plotted in arbitrary units on the abscissa  100 . The intensity is, in turn, plotted in logarithmic units on the ordinate  101 . The curve  92  shows the change in the attenuator. In this case, the attenuator is not continuously changed in a rising fashion in discrete steps. Consequently, the attenuator is varied such that firstly the intensity rises in two steps and drops again in a third step. This scheme is continued for the entire adjustment of the attenuator. As already shown in  FIG. 9 , the aperture is varied in discrete steps. The overall efficiency resulting from the combination of a varying aperture and a varying attenuator is illustrated in curve  94 . It is clearly to be seen that the intensity rises continuously in a stepwise fashion, each of the steps being equally large. It is therefore possible to speak of a quasi continuous rise in the overall efficiency. In addition, the suitable combination of the adjustment of attenuator and aperture gives rise to an overall efficiency that can be adjusted in substantially finer steps. A monotonic, exponential variation of the overall efficiency over the full range=2000:1 can be implemented by maintaining the finer graduation of the attenuator by optimized driving of the attenuator that compensates the errors arising here in the simple case ( FIG. 9 ). 
       FIG. 11  shows the resulting cutting lines  112 ,  113  and  114 , for which the aperture stop and the attenuator have been adjusted in common using the method described in  FIG. 10 . Each of the cutting lines  112 ,  113  and  114  is cut in this case with the aid of a different set of parameters from aperture stop and attenuator. All the cutting lines  112 ,  113  and  114  exhibit a continuous increase in the cutting width  115 . Each of the cutting lines  112 ,  113  and  114  exhibit no jumps in the cutting width  115  because of the adjustment of the aperture stop and of the attenuator according to the method described in  FIG. 10 . The cutting width at the end of the cutting lines  112 ,  113  or  114  is approximately 10 μm to 50 μm. Owing to the possibility of fine correction of the power values, even the use of a pinhole diaphragm instead of an iris diaphragm has proved to be definitely advantageous. An iris diaphragm has the advantage in principle that the variation of the aperture values takes place continuously, and so there is no “risk” of a laser pulse striking the space between the prescribed apertures of a pinhole diaphragm during the adjustment, and thus being blocked. By contrast, the aperture values of the pinhole diaphragm are more accurately defined, and a pinhole diaphragm can be of smaller and lighter design such that the adjustment requires only a few ms and can therefore be performed completely in the waiting time between the laser pulses. 
       FIG. 12  illustrates for example the restrictions on the possible combinations of an attenuator with dynamic range of 40:1 with an aperture stop of the same dynamic range. In this case, attenuator and aperture stop are driven simultaneously such that over a control value of 0-100 there is a strong exponential variation of the total power of 40 2 :1, that is to say 1600:1, according to the cumulative curve G. 
     In the simplest and mostly universal case, which is also implemented in  FIGS. 9 and 10 , both attenuator and aperture stop are varied in the same way in accordance with the curves of constant gradient E and F over the entire range of the control value. It follows that no special preference is accorded to a specific setting value. 
     However, it can also be rational, for example, to maintain aperture values as low as possible over a range of the control value that is as large as possible, in order to ensure a depth of field as large as possible and/or also to ensure for the cutting optics aberrations to be as slight as possible. Conversely, it can also be rational to prefer an aperture as large as possible, in order to ensure for special substrates in spatial (lateral and axial) resolution as high as possible, and at the same time to ensure as high a power density as possible at the focus (there is a disproportionate rise in the power density due to the fact that the extent of the focal spot decreases with increasing aperture!). These cases correspond to pairings of the control curves such as, for example, H and I, where in the first case l stands for the aperture and H for the attenuator, and vice versa in the second case. 
     It is found in the general case that for the purpose of driving within the scope of the prescribed object all pairs of curves (and only these!) are possible that run completely in the parallelogram A B C D from A to D and lie in pairwise fashion symmetrically in relation to the line A to D (or, in a fashion equivalent thereto: their sum yields the prescribed value G for a specific control value). 
     The control curves for attenuator and aperture are illustrated in  FIG. 13  in a fashion certainly not restricted only to profiles having monotonic variation of the curvature, but reversal points and regions of (approximately) constant value are also possible. Thus, for example, it can be required to ensure as constant an aperture as possible in accordance with the profile K over as large a range as possible about the control value of, for example, 30 (L). According to the above considerations, it is then possible to directly derive the assigned control curve J for the attenuator from the symmetry in relation to A-D or from the sum condition for G. 
     The combined driving of aperture stop A and attenuator K, particularly when use is made of the error correction according to  FIG. 10 , requires a high accuracy of the attenuator values K that are set. Unfortunately, the attenuators used typically approximately exhibit as a function of tilt angle alpha a displaced cos-shaped characteristic K=f0×cos(f1+f2×alpha)+1+f3 (f0 to f3 are individual parameters) with a maximum and a minimum, and not the desired monotonic exponential form K=g0×exp(−g1×alpha). 
     Consequently, in order to ensure accuracy for the purpose of the invention fid parameters (four or more) of an individual attenuator or, in the case of sufficiently small dispersion, the mean parameters of a production batch of attenuators are determined (see  FIG. 14 ), and are filed in the memory area of the laser head. By inverting the fid function, it is then possible to determine the associated tilt angle from a prescribed control value, and thus to “linearize” the characteristic of the attenuator (see  FIG. 15 ). It can be required, for example, in a particular case that three levels of the control value correspond with a factor of 1.25 (exponential characteristic). This results in an exponential characteristic of intensity of log 10(Intensity)∝−0.0323×control value. 
     In order to optimize the cutting speed, the calculations and movements are performed in a time “staggered” fashion, that is to say while the individual components (aperture unit  14 , attenuator unit  16 , focusing unit  19 , and deflecting unit  40  or x-y stage  2 ) are seeking a new position, or the expiry of the prescribed period for the desired laser frequency is being awaited, time-consuming calculations are already being carried out for the position respectively following thereupon. Once the feedback is then to hand that all the components have reached their desired position AND the prescribed waiting time for the laser pulse has expired, the laser trigger is released and positioning commands are transmitted at once for the following shooting position. 
     By way of example,  FIG. 16  is a schematic of a program loop for producing a cutting line, which runs as follows:
         1) Command to the wedge plates of the deflecting unit  40  to move to the last already calculated angular position (α,β);   2) Calculation of the following shooting coordinates (x,y) on the basis of the current point spacing D;   3) Conversion of these shooting coordinates into angular positions (α,β) of the wedge plates of the deflecting unit  40  (the most time consuming, as a rule);   4) Waiting until all the components have reached their desired position, AND the selected period of the laser has expired;   5) Releasing a laser trigger and resetting the counter for the period;   6) Calculating the new values for the point spacing (dx, dy), the power P, therefrom values for aperture A and attenuator K, and, if appropriate, also the focal position F;   7) Command to aperture stop, attenuator and focus motor to move to the new position;   8) Back to 1) if the end point of the cutting curve has not yet been reached.       

     In order to start the sequence, the angular positions (α,β) of the first point of the cutting curve must already have been calculated once, and aperture A, attenuator K and focal position F must already be brought into the initial position for the starting point of the cutting curve. 
     In the case of the cutting method illustrated in  FIG. 6 , it is advantageous for the purpose of the invention to monitor the position of the last laser shot at the end of the cutting line and, if appropriate, to intervene correctively. During the laser microdissection with the highest laser power the last laser pulse should always be positioned directly, before the cutting curve closes, such that as large as possible a region of the dissectate is removed in order to ensure as reliable a detachment as possible. If the last shot is set too far removed from the starting point of the cutting line, there is the risk of the dissectate being left hanging, whereas if the last point is set too close by there is the risk of the dissectate not being severed as desired but (as often observed) “turning out” from the focal plane and thus also becoming “inaccessible” for the last shot. 
     However, the total lengths of the cutting curves, which are generally fixed arbitrarily by the user, are rarely an appropriate multiple of the point spacing, particularly when the point spacing does not remain constant in the expanded modes. The substance of the invention is therefore to calculate the position, resulting from the selected cutting parameters, of this last pulse before the beginning of the cutting operation and to compel an advantageous position of the last pulse by slight variations of the parameters without thereby giving rise to an interfering discontinuity or gaps in the cutting curve or cutting line. 
     Varying the laser power parameters (aperture and attenuator) synchronously with the laser pulses delivers an extremely high cutting speed. This gives rise to positive side effects that likewise maximize the throughput in conjunction with automatic cutting of many subregions. 
     By adapting the point density to the respective laser power and the local properties of the specimen, it can be ensured that the individual laser shots produce a cut. This prevents the dissectate from being left hanging, being tilted etc., or avoids the same. An optimization of the pulse transmission can also be achieved in conjunction with severance of the region of interest. 
     The simultaneous variation of aperture and attenuator attains as large a dynamic range as possible. 
     The combined driving of attenuator and aperture stop raises the resolution in the power setting. 
     Specific properties of the specimen (within the available dynamic range) can be taken into account optimally by the variation of effective laser power via aperture and attenuator using a specific scheme. 
     The “linearization” of the attenuator K is attained by an individual calibration curve stored in the laser head. The typically cos-shaped characteristic of a tilted interference filter is thereby converted into a correctly exponential profile. 
     Furthermore, the determination of the optimum parameters for the cutting process can be performed from an image analysis either in advance in a “calibration region” of the specimen or under current, automatic optical monitoring of the cutting results during cutting. If appropriate, it is also possible to automatically recut or cut anew regions of the specimen not optimally separated. 
       FIG. 17  shows an illustration of a section from a user interface  170  via which the continuous variation of the parameters before the closure of the closed cutting line is set by means of at least one slider  171 ,  172 ,  173 . The user interface  170  is displayed to the user by the monitor  18 . The first slider  171  can be used to set the total power of the laser pulses striking the specimen. As already described, the total power results from the suitable combination of the adjustment of attenuator and aperture. The second slider  172  can be used to set the speed with which the cutting lines are to be made in the specimen. The repetition rate of the laser pulses is then set in accordance therewith. The third slider  173  can be used to set how strongly the parameters are to be changed at the end of the cutting line.