Laser microdissection methods and laser microdissection systems

Abstract

Method (200) for processing a microscopic object (20) with at least two different object regions (21-25) with different object properties by laser microdissection, wherein a laser microdissection system (100) is used with a microscope (10) having a reflected light device (76) by means of which a laser beam generated by a laser unit (70) with at least one adjustable laser beam property is guided through a microscope objective (41) of the microscope (10), wherein a user specifies a cutting line (26) on the object (20) through at least two object regions (21-25) and an impact point (27) of the laser beam on the object (20) is moved according to the cutting line (26) by means of a laser deflection device (73) in the incident light device (76) with an adjustable cutting speed, wherein at least two sections of the cutting line (26), each passing through one of the at least two object regions (21-25), are assigned at least one setting value for the at least one laser beam property and / or the cutting speed, depending on the respective object property, wherein, during the movement of the point of impact of the laser beam on the object (20) according to the cutting line (26), the at least one laser beam property and / or the cutting speed is adjusted as a function of the at least one setting value which is assigned to the section of the cutting line (26) in which the point of impact (27) is located, and wherein the object properties include a thermal sensitivity, a laser absorption coefficient, a mechanical stability, an object thickness and / or a geometric position in three-dimensional space where the laser beam hits the object (20).

Description

[0001] The present invention relates to a method for processing a microscopic object having at least two different object regions with different object properties by laser microdissection and a corresponding laser microdissection system according to the respective preambles of the independent patent claims. State of the art

[0002] Methods for processing biological samples by laser micro-dissection have existed since the mid-1970s (see e.g. ISENBERG, G. [et al.]: Cell surgery by laser micro-dissection: A preparative method. In: Journal of microscopy, Vol. 107, 1976, No. 1, pp. 19-24.) and have been continuously developed since then.

[0003] Laser microdissection allows cells, tissue regions, etc. to be isolated from an object (“sample,” “preparation”) and obtained as so-called dissectates. A particular advantage of laser microdissection is the brief contact of the object with the laser beam, which hardly alters the object. The specific extraction of the dissectate can be achieved in different ways (see, for example, STERCHI, Laser: Laser microdissection. In: Theory and practice of histological. 6th ed. London: Churchill Livingstone, 2008. pp. 575-584 – ISBN 978-0-443-10279-0, chapter “Microdissection”). For example, in known methods, a dissectate can be isolated from an object using an infrared or ultraviolet laser beam. This dissectate falls into a suitable dissectate collecting container under the influence of gravity. The dissectate can also be cut out of the object together with any adherent membrane.In laser capture microdissection, however, a thermoplastic membrane is heated using a corresponding laser beam. The membrane fuses with the desired area of ​​the specimen and can be removed by tearing it off in a subsequent step. Another alternative is to use the laser beam to attach the dissected specimen to the lid of a dissected specimen collection container. In known inverted microscope systems for laser microdissection, upwardly catapulted dissected specimens can also be attached to the bottom of a dissected specimen collection container coated with an adhesive.

[0004] The present invention is particularly applicable to methods in which a dissected specimen is removed from an object and collected in a dissected specimen collection container located underneath. The invention is particularly suitable for contactless collection systems for dissected specimens.

[0005] Known microscope systems for laser microdissection, such as those known from WO 98 / 14816 A1, have a reflected light device into whose beam path a laser beam is coupled. The laser beam is focused by the respective microscope objective onto the object resting on a motor-driven, automatically movable microscope stage. A cutting line is generated by moving the microscope stage during cutting in order to move the object relative to the fixed laser beam. However, this has the disadvantage, among other things, that the object cannot be easily viewed while the cutting line is being generated, as it moves in the field of view, and the image may appear blurred.

[0006] Therefore, laser microdissection systems that have laser deflection or laser scanning devices configured to move the laser beam or its point of incidence over a stationary object are more advantageous. Such laser microdissection systems, which also offer particular advantages within the scope of the present invention, are explained in detail below. A particularly advantageous laser microdissection system that has a laser deflection device with mutually adjustable glass wedges in the laser beam path is described, for example, in EP 1 276 586 B1.

[0007] In both cases—in laser microdissection systems where the microscope stage is moved, and in laser microdissection systems that feature a laser deflection device—pulsed lasers are generally used, with each laser pulse creating a hole or depression in the specimen. A cutting line is created by a series of such holes or depressions, possibly with overlap.

[0008] Document DE 10 2004 023 262 A1 discloses a method in which, during automatic image evaluation, several regions are automatically selected and the geometric parameters of each selected region are stored. Control signals for automatically cutting the mass and / or catapulting the object can then be generated based on the stored geometric parameters of the selected regions.

[0009] From document DE 10 2004 051 508 A1 a method is known in which, through the additional use of microscopic and / or camera-based contrasting methods by an imaging method, e.g. by means of a camera, a specimen section can be presented in such a way that the laser cutting line can be automatically determined on the basis of this image using image analysis.

[0010] Further laser microdissection systems and methods for laser microdissection are disclosed in the documents DE 10 2006 009 564 A1, DE 10 2011 001 474 A1, DE 10 2007 016 301 A1 and DE 10 152 404 A1.

[0011] Laser microdissection can be used to obtain single cells or defined tissue regions, which are then subjected to various diagnostic analysis procedures. In oncology, for example, laser microdissection can be used to specifically isolate tumor cells from a microscopic section and examine them for specific metabolites or proteins. It is important to ensure that the relevant material is obtained as gently as possible and, in particular, that there is no thermal deterioration.

[0012] On the other hand, the corresponding individual cells or tissue regions should also be obtained as easily and as effortlessly as possible for the user. This proves to be particularly problematic when a corresponding object has several different object regions with different object properties that influence their processability with the laser beam. For example, an object region of interest may be surrounded by regions that are difficult to transect using the laser beam. For example, while a first region has already been completely transected by the laser beam, another region requires complex reworking to fully isolate the region of interest. This is time-consuming and potentially error-prone.In particular, there is a risk of subjecting the object as a whole to too intensive processing, which entails thermal stress that may render the region of interest unusable for subsequent analyses.

[0013] There is therefore a need for improved possibilities for processing microscopic objects with at least two different object regions with different object properties by laser microdissection. Disclosure of the invention

[0014] The present invention proposes a method for processing a microscopic object having at least two different object regions with different object properties by laser microdissection and a corresponding laser microdissection system having the features of the independent patent claims. Preferred embodiments are the subject of the dependent patent claims and the following description.

[0015] The present invention is based on a method known per se for processing a microscopic object with at least two different object regions with different object properties by laser microdissection. As explained above, such a method advantageously uses a laser microdissection system that comprises a microscope with a reflected light device. By means of the reflected light device, as also explained with reference to Fig. As explained in Figure 1, a laser beam generated by a laser unit with at least one adjustable laser beam property is guided through a microscope objective of the microscope. A user specifies a cutting line on the object, which, in the method according to the invention, runs through the at least two object regions. This occurs, for example, in a specification step preceding the actual processing.

[0016] During the actual processing of the microscopic object, i.e., in a processing step, the point of incidence of the laser beam on the object is shifted by means of a laser deflection device in the aforementioned incident light device according to the previously specified cutting line, i.e., according to the cutting line that runs through at least two different object regions. The shifting occurs at an adjustable cutting speed.

[0017] Such a cutting speed can, in particular, be a continuous feed rate of the point of impact of the laser beam on the microscopic object. However, in the context of the present application, a "cutting speed" is also understood to mean the speed at which a corresponding point of impact is moved overall. For example, known methods for laser microdissection provide for the emission of several laser pulses at each point of impact of a laser beam on an object. Between corresponding steps, the point of impact of the laser beam is shifted. In such cases, the point of impact of the laser beam on the object is shifted step by step, whereby the cutting speed here corresponds to the average speed of such a shift.

[0018] The invention is therefore based on a known laser microdissection system. Such a laser microdissection system comprises a microscope having a reflected light device, a microscope objective, and a laser unit. A beam path of a laser beam from the laser unit passes through the reflected light device and the microscope objective and intersects an object plane of the microscope objective at an adjustable intersection point, which is specified by means of control signals to the laser deflection device.

[0019] The invention is particularly used in so-called non-contact laser microdissection systems. These are characterized by the fact that a dissected specimen is not attached to membranes or the like, but rather falls downwards from the specimen due to gravity. The specimen is mounted in upright systems on the underside of a slide and is processed through the slide from the top side.

[0020] Using the incident light device, a laser beam from a laser light source is coupled into the microscope's observation beam path. The laser beam is focused onto the object through the microscope objective, which is also used for viewing the object.

[0021] To avoid any misunderstandings, it should be emphasized at this point that the laser microdissection system employed within the scope of the invention is used with objects that are already prepared for microscopy. These may, for example, be thin sections cut from a larger tissue block using a microtome. Such a tissue block may, for example, be a fixed organ or a biopsy of a corresponding organ. The laser microdissection system according to the invention is therefore not used to obtain objects, but rather to process them and to isolate specific areas thereof. It is understood that the invention can also be used with objects that are not obtained using a microtome, e.g., smears, macerates, etc.

[0022] Microtomes are used exclusively for the preparation of microscopic specimens. Microtomes can also incorporate lasers for this purpose. The sections obtained using a microtome are applied to a slide, as mentioned above, where appropriate, and stained, etc. Only then are they available for use in the laser microdissection system. A microtome differs fundamentally from a laser microdissection system in that, among other things, the latter produces sections with the most homogeneous cutting thickness possible. Microtomes are therefore designed to produce a large number of identical sections with parallel cutting surfaces, whereas laser microdissection systems are configured to separate dissected specimens according to object-specific criteria, such as visual criteria. Therefore, a person skilled in the art would not transfer the technical solutions used in microtomes to laser microdissection systems.

[0023] Microtomes also do not include microscopes in which a laser beam is coupled into the observation beam path. Therefore, in microtomes, the laser beam is never focused onto an object, such as a tissue block, through a microscope objective that is also used for observation.

[0024] In laser microdissection systems with a laser deflection device, as used according to the invention, the microscope stage is arranged fixedly relative to the microscope objective with respect to the x- and y-direction (i.e. perpendicular to the optical axis of the microscope objective) when separating the dissected material, i.e. during the dissecting process.

[0025] In contrast to laser microdissection systems with a motor-driven microscope stage (scanning stage) during the dissection process, which must have a high positioning accuracy, especially with high magnification objectives, in order to enable precise sections, laser microdissection systems with a laser deflection device prove to be simpler and more cost-effective to manufacture and have precision advantages.

[0026] In a particularly advantageous embodiment, the laser deflection device comprises two thick glass wedge plates ("glass wedges") that are inclined relative to an optical axis and independently rotatable about an optical axis. These wedge plates generate a beam deflection through their wedge angles. By rotating the glass wedge plates, the resulting deflection angle of the laser beam relative to the optical axis is variable. At the output of the laser deflection device, the laser beam exhibits a lateral beam offset relative to the optical axis due to the thickness and inclination of the glass wedge plates, and strikes the center of the objective pupil of the microscope objective for all deflection angles. The intersection point of the laser beam with the object plane is thus adjustable.

[0027] Such a laser deflection device is particularly advantageous over other laser deflection devices such as mirror scanners, galvanometer scanners, or stepper motor scanners because it does not have to be arranged in a plane conjugate to the objective pupil. This also eliminates the need for pupil imaging to ensure that the deflected beam hits the objective pupil. For microdissection with UV laser light, for example, a UV-compatible pupil imaging device would be required. Further advantages of such a laser deflection device with wedge plates are mentioned, for example, in EP 1 276 586 B1. Advantages of the invention

[0028] According to the invention, during the displacement of the point of impact of the laser beam on the object, the at least one laser beam property and / or the cutting speed (which, as explained above, can also be an average speed in the case of a step-by-step displacement) is adjusted depending on the object property of the object region of the at least two object regions in which the point of impact is (currently) located. As explained above, within the scope of the method according to the invention, a cutting line on the object is specified through the at least two object regions. During processing, the point of impact of the laser beam is thus also guided through these at least two object regions. At any given time, the point of impact of the laser beam is therefore in one of the at least two different object regions, and a corresponding adjustment is made.

[0029] According to the invention, at least two sections of the cutting line, each of which runs through one of the at least two object regions, are assigned at least one setting value for the at least one laser beam property and / or the cutting speed depending on the respective object property, and that during the displacement of the point of impact of the laser beam on the object according to the cutting line, the at least one laser beam property and / or the cutting speed is adjusted depending on the at least one setting value that is assigned to the section of the cutting line in which the point of impact is located.

[0030] Such object properties include thermal sensitivity, laser absorption coefficient, mechanical stability, object thickness and / or a geometric position in three-dimensional space at which the laser beam impinges on the object.

[0031] Adjusting at least one laser beam property and / or the cutting speed depending on the thermal sensitivity of an object region makes it possible to reduce the thermal load on the corresponding object and thus undesirable effects. For example, the invention can provide for the use of comparatively low laser energy and / or a high cutting speed in object regions with high thermal sensitivity. This keeps the thermal load on a corresponding sample region low. If, in such a case, the object is not completely severed in a corresponding object area as desired, multiple passes can be provided, which, however, allows the sample to cool between these passes.

[0032] In addition to thermal sensitivity, as explained, a laser absorption coefficient of an object region can also be taken into account in order to adjust at least one laser beam property and / or the cutting speed based on this. Areas with a comparatively high laser absorption coefficient absorb a larger proportion of the laser energy, so that, for example, a higher cutting speed and / or a lower number of laser pulses or lower laser energy are required for their processing. Such areas can thus be cut more gently if gentler laser beam properties are used.

[0033] Conversely, object regions with high mechanical stability, i.e., hard, difficult-to-cut object regions, may require higher laser power and / or a higher number of laser pulses. The cutting speed is reduced in such areas, for example.

[0034] Furthermore, for example, object regions with a greater object thickness require more laser energy for cutting and / or only allow a low cutting speed.

[0035] Particular advantages are achieved when the present invention is used in a method in which three-dimensional microscopic objects are sectioned. A "three-dimensional" microscopic object is understood here to be an object that, unlike conventional microscopic thin sections, does not have a vertical extension that is negligible compared to the horizontal extension. Such three-dimensional microscopic objects are therefore taller than conventional microscopic thin sections.

[0036] For processing corresponding three-dimensional objects using laser microdissection, a laser microdissection system is known, for example, from the as yet unpublished German patent application 10 2012 207 240.3, which comprises a z-adjustment device for the microscope stage to move it in the z-direction and / or an optical focus shifting device to move the focus of the laser beam in the z-direction. In such a laser microdissection system, this adjustment device(s) can be controlled together with a laser deflection device such that the focus of the laser beam can be moved along a three-dimensional cutting line in the x-, y-, and z-directions. This allows a corresponding laser beam to impinge on a corresponding object at different geometric positions. Such a position is advantageously also considered as an object property within the scope of the present invention.

[0037] Within the scope of the present invention, the adjustment of different laser beam properties can be advantageous. Such laser beam properties can include at least a laser energy or laser power, a laser aperture, and / or a laser frequency of the laser beam.

[0038] The laser energy setting directly influences the amount of energy delivered at each point of impact of the laser beam. The same applies to the cutting speed, whereby at a slower cutting speed, more energy is delivered per distance than at a faster cutting speed - provided the laser energy, the laser frequency, and / or the laser aperture are not changed. The same applies to the laser frequency. The laser aperture also directly influences the amount of energy delivered per area. At least two such laser beam properties can also be changed synchronously if the laser beam properties are to be adjusted depending on the respective object property. By adjusting the aforementioned laser beam properties, the expert has a multitude of options for influencing the means available for processing a microscopic object.

[0039] It is particularly advantageous if the object properties of at least two object regions are already specified when the cutting line is defined on the object. This can be done, for example, when a user defines a corresponding cutting line.

[0040] Such a cutting line is specified, for example, on a computer screen using familiar input devices, such as a mouse, a tablet, and / or other input devices. The specification is typically made using a microscopic image of the object to be processed. A corresponding image has therefore already been taken in advance.

[0041] This makes it comparatively easy to (visually and / or automatically) recognize the different object regions and to assign corresponding object properties to these object regions. For example, a user has knowledge of the existing object properties due to their experience in processing microscopic objects by laser microdissection. A user can, for example, assign corresponding object properties to a cutting line by marking certain areas of this cutting line in a suitable way, e.g. with color and / or by assigning numerical values. The assignment can also be done in an abstract way or intuitively. For example, the user can specify a thicker cutting line in object regions that require more intensive laser processing (i.e., for example, with higher laser energy and / or slower cutting speed).This can be achieved, for example, by firmly pressing down with a tablet stylus. Advantageously, the object properties of at least two object regions are stored together with the cutting line and / or transferred to a corresponding laser microdissection system, for example, from a corresponding computer.

[0042] It is particularly advantageous if the laser microdissection system is enabled to automatically determine the object properties of at least two object regions. As already explained above, the specification of a cutting line on an object in conventional laser microdissection systems typically takes place on a computer screen. In this case, corresponding images are available, which can be evaluated using an image evaluation unit of a laser microdissection system. The image evaluation unit can, for example, determine an object property based on different colors, gray values, densities, structural features and / or based on a determined spatial position of object regions or points in such object regions. For example, an optically dense object region can indicate that a corresponding object requires intensive processing, so that the laser energy can be increased at such a location.

[0043] Advantageously, as explained, the object properties of the at least two object regions are each assigned to specific areas of the cutting line. The cutting line can also be transmitted to the laser microdissection system separately from the object image, provided that the system is calibrated with a corresponding imaging system and enables reliable processing of a corresponding sample even without an associated optical image.

[0044] The method according to the invention can be advantageously used if the point of incidence of the laser beam on the object is shifted at least in or parallel to an object plane of the microscope objective. The object plane of the microscope objective, which advantageously represents a flat surface, defines the x- and y-planes in known laser microdissection systems, which, for example, also corresponds to the surface of the microscope stage. A microscopic object is arranged in this xy-plane and can be shifted accordingly. The laser deflection device used can shift the point of incidence of the laser beam on the object in this plane or parallel to it.

[0045] However, particular advantages arise when the point of impact of the laser beam on the object is shifted in a further direction, namely in the z-direction perpendicular to the object plane or a plane parallel to it. This is the case, for example, in the aforementioned German patent application 10 2012 207 240.3. Here, too, the method according to the invention enables adjustment of the laser beam properties and / or the cutting speed depending on at least one object property.

[0046] A laser microdissection system with a microscope having an incident light device for guiding a laser beam generated by a laser unit with at least one adjustable laser beam property through a microscope objective of the microscope is also subject of the present invention. The laser microdissection system has means for a user to specify a cutting line on the object through the at least two different object regions with different object properties. The laser microdissection system has a laser deflection device in the incident light device for shifting the point of incidence of the laser beam on the object according to the cutting line at an adjustable cutting speed.According to the invention, such a laser microdissection system comprises means for assigning at least one setting value for the at least one laser beam property and / or the cutting speed to at least two sections of the cutting line, each of which runs through one of the at least two object regions, depending on the respective object property, and adjustment means for adjusting the at least one laser beam property and / or the cutting speed at which the point of impact of the laser beam on the object is shifted according to the cutting line. The adjustment means are configured to adjust the at least one laser beam property and / or the cutting speed depending on the at least one setting value assigned to the section of the cutting line in which the point of impact is located during the shifting of the point of impact of the laser beam on the object.

[0047] Such a laser microdissection system is particularly designed to perform a method as previously described. Such a laser microdissection system therefore benefits from the advantages previously described.

[0048] In particular, such a laser microdissection system comprises detection means for detecting the object properties of the at least two different object regions. These can, for example, be integrated into an image evaluation unit that captures an image of the microscopic object and automatically detects the corresponding object regions or object properties.

[0049] Embodiments of the invention are explained in more detail below with reference to the accompanying drawings. Short description of the drawings Fig. 1 shows a laser microdissection system that can be used in the context of a method according to the invention, in a schematic representation. Fig. 2 illustrates the specification of a cutting line according to an embodiment of the invention in a schematic representation. Fig. 3 illustrates a method according to a preferred embodiment of the invention in the form of a schematic flow chart.

[0050] In the figures, corresponding elements are indicated by identical reference numerals and will not be explained repeatedly. Detailed description of the drawings

[0051] In Fig. 1, a laser microdissection system that can be used to carry out a method according to the invention is schematically illustrated and designated overall by 100. The laser microdissection system 100 corresponds in essential parts to that disclosed in EP 1 276 586 B1, to which reference is expressly made here. A coordinate system, by means of which the axes or directions x, y and z mentioned below are illustrated, is shown in Fig. 1 designated 110.

[0052] The laser microdissection system 100 comprises a microscope 10. An illumination device 12, only partially shown here, can be provided in a microscope base 11 of the microscope 10. This can, for example, comprise a light source (not shown) and suitable means for influencing the illumination light provided by the light source, for example, filters and / or apertures. A condenser unit 90 can be provided for transmitted light illumination and for setting suitable contrast or observation methods.

[0053] For example, a user input and / or user information unit 13 can also be arranged on the microscope base 11, which can be designed as a touchscreen, for example, and via which the user can enter and / or read out viewing and / or processing parameters, for example.

[0054] Furthermore, a drive knob 14 is provided. This serves to operate a coarse and a fine drive for adjusting the height of a microscope stage 30. An object located on a slide (not labeled here) on a slide holder 50, for example, a section applied to the slide, can thereby be brought into an object plane 51 of an objective 41. The objective 41 is mounted alongside other objectives 42 in an objective turret 40. A protective hood 15 can be provided to protect against laser radiation.

[0055] Observation light emanating from the object in the object plane 51 travels along an observation beam path a. In a tube unit 60 with suitable output devices 61, a preferably variable portion of the observation light, for example, by 60°, can be output and presented to a user via a pair of eyepieces 62. A further portion of the observation light can be input into a digital image acquisition unit 63 and captured as an image. An image evaluation unit 64 can be assigned to the image acquisition unit 63, locally, in a control unit 82 or a control computer 81 (see below), or in another spatial arrangement.

[0056] The laser microdissection system 100 comprises a laser unit 70 with a laser light source 75. A laser beam b provided by the laser light source 75, which may be, for example, a UV laser light source, is deflected in a reflected light unit, designated here as a whole by 76, at a first deflection mirror 71 and a second deflection mirror 72 and focused by the objective lens 41 onto the object in the object plane 51.

[0057] In the laser microdissection system 100, the location at which the laser beam b impinges on the object in the object plane 51 can fundamentally be adjusted in different ways. On the one hand, a manual adjustment device 31 can be provided, by means of which the microscope stage 30, designed as a cross-stage, can be adjusted in the x- and y-directions (i.e., here perpendicular or parallel to the paper plane). In addition to the adjustment device 31, electromechanical actuating means can also be provided, which can be controlled, for example, by a control unit 82 or whose position can be detected by the control unit 82.

[0058] The control unit 82 can also control any other motorized functions of the laser microdissection system 100 and in particular provide an interface to an external control computer 81, which can be connected via corresponding connections 83.

[0059] For laser microdissection, a laser deflection device 73 can be provided, in particular. By means of the laser deflection device 73, the laser beam b can be deflected relative to an optical axis c running between the first deflection mirror 71 and the second deflection mirror 72. The laser beam can therefore impinge on the second deflection mirror 72, which can be designed, for example, as a dichroic splitter, at different positions and is thus also focused on the object in the object plane 51 at different positions. Deflection by means of a laser deflection device 73 is shown in detail in EP 1 276 586 B1. It should be emphasized that different options for deflecting a laser beam b or for positioning the object in the object plane 51 relative to the laser beam b can be used here. The invention is not limited to the example shown.

[0060] In the example shown, the laser deflection device 73 has two solid glass wedge plates 731, which are inclined relative to the optical axis c and can be rotated independently of one another about the optical axis c. For this purpose, the wedge plates 731 are mounted on ball bearings 732. Each of the wedge plates is connected to a gear 733. The gears 733 can each be rotated by actuators 734, which can be supplied with corresponding control signals and drive the gears 733 accordingly. The actuators 734 can have position sensors 735 (shown here only on the right-hand actuator 734). A position detected by the position sensors 735 can be transmitted to the control unit 82.

[0061] The control computer 81 and / or the control unit 80 can also be configured to detect object regions with different object properties. For example, a cutting line, as shown in the following Fig. 2. The control computer 81 and / or the control unit 80 can also be configured to automatically detect a cutting line, as is generally known. This can be done, for example, based on an image analysis in the image analysis module 64.

[0062] In Fig. 2 schematically shows the specification of a cutting line for processing a microscopic object by laser microdissection according to an embodiment of the invention.

[0063] In the illustrated example, the microscopic object is designated overall by 20. It comprises five different object regions, designated here by 21 to 25, which exhibit at least partially different object properties, as previously explained. The object 20 is mounted on a slide 29 and secured accordingly. In the illustrated example, the object 20 is a flat thin section; however, as explained, the method according to the invention can also be used for three-dimensional objects.

[0064] In the Fig. 2A to 2C of the Fig. 2 are the slide 29 with the object 20 ( Fig. 2A), the slide 29 with the object 20 and a cutting line 26 ( Fig. 2B) and the slide 29 with the object 20, the section line 26 and different sections of the section line 26, which correspond to different object properties of the object regions 21 to 25 of the object 20.

[0065] In the example shown, for example, the object region 25 is to be separated from the object 20 using laser microdissection. However, the object region 25 is surrounded by the object regions 21 to 24, which may have different object properties. For example, the object region 23 represents a region that is difficult to cut through and requires particularly intensive processing using a laser beam due to its mechanical properties, for example. In contrast, the object region 21, for example, represents a sensitive and easy-to-cut object region. If an attempt were made to separate the object region 25 from the object 20 using uniform laser beam properties and / or a uniform cutting speed, the object region 25 might still adhere to the object region 23 while already being separated from the object region 21.

[0066] Therefore, as in Fig. 2B, a cutting line 26 is first specified around the object region 25. The cutting line 26 can, for example, be specified at a defined distance from the object region 25. A user can also, for example, mark the object region 25, whereupon a laser microdissection system automatically defines the cutting line 26 at a certain safety distance from the object region 25. Specifying or defining a cutting line 26 can involve varying degrees of automation. Certain laser microdissection systems are configured for the fully automatic detection of object regions, so that the user only needs to, for example, mark the center point of the object region 25, so that a cutting line 26 is automatically specified around this object region 25. The object regions 21 to 25 themselves can also be detected automatically.

[0067] As in Fig. 2C, different sections of the cutting line 26 are defined in a next step. The definition can, for example, be based on the object properties of the object regions 21 to 25 automatically detected in a previous step. Alternatively, a user can also mark different sections of the cutting line 26 and manually assign different laser beam properties and / or cutting speeds to them. This is shown in the Fig. 2C by different dashed lines on the cutting line 26. Here, too, a corresponding cutting line can be automatically subdivided into the aforementioned sections. The point of impact of a laser beam on the object 20, illustrated here as 27, always lies in one of the defined object regions 21 to 25, to which corresponding laser beam properties and / or cutting speeds are assigned.

[0068] When processing the sample 20 following the definition of the cutting line 26 and the division of the cutting line 26 into the different sections, the laser beam properties and / or the cutting speed can therefore be adjusted accordingly.

[0069] Advantageously, the cutting line 26 is transmitted to a laser microdissection system or a control unit provided therefor with the laser beam properties and / or cutting speeds defined according to the different sections.

[0070] In Fig. 3, a method according to an embodiment of the invention is shown in the form of a schematic flow chart and is designated overall by 200. The method begins with a step 201 in which, for example, an object 20 or a corresponding object carrier 29 (cf. Fig. 2) into a laser microdissection system, for example the laser microdissection system 100, which is Fig. 1 is shown.

[0071] In a step 202, an image of the object 20 or the slide 29 with the object 20 located thereon is generated and transmitted, for example, to a computer 81 or a control unit 82 of the laser microdissection system. In step 202, the different object regions of the object 20 can also be automatically detected and also transmitted to the computer 81 and the control unit 82. In a step 203, a corresponding computer 81 or a control unit 82 receives the relevant data.

[0072] In a step 204, a user specifies a cutting line, for example, on a screen of the computer 81 and / or via a user input unit 13 of a laser microdissection system connected to a control unit 82. Alternatively, a corresponding cutting line can also be determined automatically. In parallel, the user selects specific areas of the cutting line located in different sample regions in order to set different laser beam properties and / or cutting speeds in these regions. However, as explained, this can also be done fully automatically.

[0073] The corresponding data, ie, the cutting lines with the laser beam properties and / or cutting speeds assigned to them in sections, are transmitted, for example, to a control unit 82 of the laser microdissection system 100. Based on this, control signals for the laser deflection unit 73 and / or the laser unit 70 are generated.

[0074] This occurs in step 205, which corresponds to the actual processing step of the object to be processed by laser microdissection. In step 205, the object 20 is cut according to the cutting line and the laser beam properties and / or cutting speeds defined for each section.

[0075] The method ends in step 206, in which a corresponding dissection specimen is available.

Claims

[1] Method (200) for processing a microscopic object (20) with at least two different object regions (21-25) with different object properties by laser microdissection, wherein a laser microdissection system (100) is used with a microscope (10) having a reflected light device (76), by means of which a laser beam generated by a laser unit (70) with at least one adjustable laser beam property is guided through a microscope objective (41) of the microscope (10), wherein a cutting line (26) on the object (20) through the at least two object regions (21-25) is specified by a user and an impact point (27) of the laser beam on the object (20) is shifted according to the cutting line (26) by means of a laser deflection device (73) in the incident light device (76) at an adjustable cutting speed, wherein at least two sections of the cutting line (26), each passing through one of the at least two object regions (21-25), are assigned at least one setting value for the at least one laser beam property and / or the cutting speed depending on the respective object property, wherein, during the displacement of the point of impact of the laser beam on the object (20) according to the cutting line (26), the at least one laser beam property and / or the cutting speed is adjusted as a function of the at least one setting value assigned to the section of the cutting line (26) in which the point of impact (27) is located, and wherein the object properties include a thermal sensitivity, a laser absorption coefficient, a mechanical stability, an object thickness and / or a geometric position in three-dimensional space at which the laser beam impinges on the object (20). [2] The method (200) of claim 1, wherein the at least one laser beam property comprises at least one of a laser energy, a laser aperture, and a laser frequency of the laser beam. [3] Method (200) according to one of the preceding claims, in which the object properties of the at least two object regions (21-25) are specified when specifying the cutting line (26) on the object (20). [4] Method (200) according to one of the preceding claims, wherein the object properties of the at least two object regions are determined by means of the laser microdissection system (100). [5] Method (200) according to one of the preceding claims, in which the object properties of the at least two object regions (21-25) are each assigned by a user to the areas of the intersection line (26). [6] Method (200) according to one of the preceding claims, in which the point of incidence (27) of the laser beam on the object (20) is displaced at least in or parallel to an object plane (51) of the microscope objective (41). [7] Method (200) according to claim 6, wherein the point of incidence (27) of the laser beam on the object (20) is further shifted perpendicular to the object plane (51) of the microscope objective (41) or a plane parallel thereto. [8] Laser microdissection system (100) comprising: a microscope (10) with an incident light device (76) for guiding a laser beam generated by a laser unit (70) with at least one adjustable laser beam property through a microscope objective (41) of the microscope (10), Means (81) for specifying a cutting line (26) on an object (20) through at least two different object regions (21-25) with different object properties, a laser deflection device (73) in the incident light device (76) for shifting an impact point (27) of the laser beam on the object (20) according to the cutting line (26) and at an adjustable cutting speed, Means for assigning at least one setting value for the at least one laser beam property and / or the cutting speed to at least two sections of the cutting line (26), each of which runs through one of the at least two object regions (21-25), depending on the respective object property, and Adjustment means (82) for adjusting the at least one laser beam property and / or the cutting speed as a function of the at least one setting value assigned to the section of the cutting line (26) in which the point of impact (27) is located during the displacement of the point of impact of the laser beam on the object (20), wherein the object properties include a thermal sensitivity, a laser absorption coefficient, a mechanical stability, an object thickness and / or a geometric position in three-dimensional space at which the laser beam impinges on the object (20). [9] Laser microdissection system (100) according to claim 8, comprising detection means for detecting the object properties of the at least two different object regions (21-25).

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