DE102014203747A1 - Laser microdissection system and laser microdissection method - Google Patents

Abstract

A laser microdissection system (100) comprising a microscope (10) having a receptacle (52) for receiving a biological sample (51) and a Auflichteinrichtung (76) with a laser deflector (73) which a provided by a laser unit (70) laser beam (70). 77) through a microscope objective (41) of the microscope (10) leads to a sample area (510) of the biological sample (51) and which shifts a point of incidence of the laser beam (77) on the sample area (510), the laser microdissection system (100) at least a further laser deflection device (73 ', 73' ') for guiding at least one further laser beam (77', 77 '') on the sample area (510) or on at least one other sample area (530, 540) and for displacing the point of impact of the at least one further laser beam (77 ', 77' ') on the respective sample area.

Description

The present invention relates to a laser microdissection system and a laser microdissection method according to the respective preambles of the independent claims as well as various uses of the claimed laser microdissection system.

State of the art

Methods for processing biological samples by laser microdissection have existed since the mid-1970s and have been continuously developed since then. In laser microdissection, cells, tissue regions, etc., can be isolated from a biological sample ("object") and recovered as so-called dissectates. A particular advantage of laser microdissection is the short contact of the sample with the laser beam, by which it is hardly changed. The recovery of the dissectates can be done in different ways.

For example, in known methods, a dissectate can be isolated from a sample by means of an infrared or ultraviolet laser beam which falls under the influence of gravity into a suitable dissektate collection container. The dissectate can be cut out of the sample also together with a membrane attached to the sample. In the so-called "laser capture microdissection", however, a thermoplastic membrane is heated by means of a corresponding laser beam. The membrane fuses with the desired area of the sample and can be removed by tearing in a subsequent step. Another alternative is to attach the dissectate by means of the laser beam to a lid of a Dissektatauffangbehälters. In known inverse microscopy systems for laser microdissection, upwardly transported dissectates may also be attached to the bottom of a dissectate collection container provided with an adhesive coating.

Known microscope systems for laser microdissection have a Auflichteinrichtung, in the beam path of a laser beam is coupled. The laser beam is focused by the particular microscope objective used on the sample, which rests on a motor-automatically movable microscope stage. A cutting line can be created by moving the microscope stage during cutting to move the sample relative to the fixed laser beam. However, this has, inter alia, the disadvantage that the sample can not be readily observed during the generation of the cutting line, since the sample moves in the field of vision and the image appears blurred or blurred without further compensation measures.

Therefore, laser microdissection systems which have laser deflection or laser scanning devices which are set up to move the laser beam or its point of impact on the stationary sample to be dissected are more advantageous. Such laser microdissection systems, which are also to be used in the context of the present invention and offer special advantages there, are explained in detail below. A particularly advantageous laser microscope system which has a laser deflection device with mutually adjustable glass wedges in the laser beam path, is for example in the EP 1 276 586 B1 described.

In both cases, that is to say in systems with a moving or stationary sample, pulsed lasers are generally used, a hole or depression being produced in the sample by each laser pulse. A cutting line is created by a friction of such holes or depressions, optionally with overlap.

The laser microdissection can be used to obtain single cells or defined areas of tissue, which are separated by a laser beam from the surrounding tissue and subsequently subjected to different diagnostic analysis methods, for example. In oncology, for example, laser microdissection can be used to specifically isolate tumor cells from a microscopic section and to examine specific metabolites or proteins.

Furthermore, the laser microdissection can also be used for the manipulation of single cells or defined tissue areas. Here, for example, to think of a fluorescence excitation or to use as optical tweezers.

One problem with laser microdissection techniques is the speed limitation of cutting out a dissectate or manipulating a target region on a sample having multiple sample sections to be trimmed. Such sample sections are processed sequentially.

In this so-called serial section cutting (SSC), various serial sections, which originate from one and the same preparation, are processed in parallel. Such a method is for example from the US 2012/0045790 A1 known.

According to this US 2012/0045790 A1 are taken from a preparation two in particular adjacent sample sections and each on applied a slide. The first sample section is on a standard slide (with coverslip), the second sample section is on a so-called dissection slide (without cover glass). A sample area on the standard slide is examined microscopically, specifying a sample section of interest. The image of the first sample section is superimposed with the image of the second sample section, so that the sample region of interest is projected from a first sample section onto the corresponding sample region in the second sample section. This corresponding sample area can now be cut out by means of laser microdissection from the second sample section and fed to a further analysis. Even with this laser microdissection method, several sample regions of interest can only be processed sequentially.

It is therefore an object of the present invention to accelerate the process of laser microdissection, wherein, in particular, a flexibilization of this process is to be made possible at the same time.

Disclosure of the invention

To achieve the stated object, the present invention proposes a laser microdissection system, uses of this system and corresponding methods for laser microdissection according to the independent patent claims. Advantageous embodiments are the subject of the respective subclaims and the following description.

The present invention is based on a laser microdissection system known per se with a microscope which has a recording device for receiving a biological sample and an incident light device with a laser deflection device, which guides a laser beam provided by a laser unit through a microscope objective of the microscope onto a sample region of the biological sample and which shifts a point of impact of the laser beam on the sample area.

A corresponding laser microdissection system is described in detail below with reference to FIGS 1 explained. By means of the incident light device, the laser beam from a laser light source is coupled into the observation beam path of the microscope in such a laser microdissection system. The laser beam is focused through the microscope objective, which is also used to view the sample. Thus, in other words, runs the beam path of the laser beam of the laser unit through the Auflichteinrichtung and through the microscope objective and intersects an object plane of the microscope objective at an adjustable intersection, which is predetermined by means of control signals to the laser deflection.

To avoid misunderstandings, it should be emphasized here that the laser microdissection system used in the context of the present invention is used with samples which are already ready for microscopy and laser dissociable (ie without coverslip or similar cover, but not liquid-coated). These may be, for example, tissue thin sections which have been separated out of a larger tissue block by means of a microtome, but in the present case also thicker sections from a corresponding tissue block. Such a tissue block may be, for example, a fixed organ or a biopsy of a corresponding organ. The laser microdissection system according to the invention therefore does not serve to obtain samples but to process them and to isolate certain areas thereof. It is understood that the present invention may also be used with samples that are not obtained by means of a microtome, e.g. with smears, macerates, etc. As mentioned, however, the invention is also suitable for processing thicker samples which have not been prepared by means of a microtome.

Microtomes are used exclusively in the preparation of microscopic samples. Microtomes can also have lasers for this purpose. The sections obtained by means of a microtome are applied to a microscope slide as mentioned above, optionally attached there, stained, etc. Only then are these available for use in the laser microdissection system. Among other things, a microtome differs fundamentally in its operation from a laser microdissection system in that it produces cuts with as homogeneous a cutting thickness as possible. Microtomes are therefore designed to generate a large number of identical cuts with parallel cut surfaces, whereas laser microdissection systems are arranged to separate dissectates according to sample-dependent criteria, for example according to visual morphological criteria. In the present case, the laser microdissection system is used in particular for separating out sample particles, which are subsequently taken up in a suspending fluid. The person skilled in the art would therefore not transfer technical solutions used in microtomes to such laser microdissection systems due to the completely different objectives.

In order to separate out sample particles or sample areas, that is to say to obtain dissectates, a sample area is completely severed along a section line and thus separated from the replaced the surrounding sample. For example, it is not possible to isolate material from different superimposed layers of tissue separately. Therefore, a dissectate obtained by laser microdissection always represents a "bulk sample area" throughout the entire thickness of the processed sample; a differentiation into individual tissue layers by means of laser microdissection is not possible or can only take place indirectly over the thickness of the sample area.

The microscope stage is fixedly arranged in laser microdissection systems with a laser deflection device, as used in accordance with the invention, when the sample is processed relative to the microscope objective with respect to the x and y directions (ie perpendicular to the optical axis of the microscope objective).

In contrast to laser microdissection systems with a microscope stage (scanning stage) moved by a motor during the dissecting process, which has to have a high positioning accuracy, in particular in the case of high-magnification objectives, in order to allow precise cuts, laser microdissection systems with a laser deflector are simpler and more cost-effective to produce and have precision advantages ,

In a particularly advantageous embodiment, the laser deflection device has two thick glass wedge plates ("glass wedges") which are inclined relative to an optical axis and can rotate independently of one another about an optical axis, which beam deflections are produced by their wedge angles. Due to the rotation of 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 has a lateral beam offset with respect to the optical axis due to the thickness and the oblique position of the glass wedge plates and strikes the center of the objective pupil of the microscope objective for all deflection angles. The intersection of the laser beam with the object plane is thus adjustable.

Such a laser deflection device is therefore particularly advantageous over other laser deflection devices such as mirror scanners, galvanometer scanners or stepper motor scanners because they do not have to be arranged in a plane conjugate to the objective pupil. Thus, no so-called pupil imaging is required in order to achieve that the deflected beam hits the objective pupil. In the case of laser microdissection with UV laser light, for example, a UV-compatible pupil image would be required. Further advantages of such a laser deflection device with wedge plates are for example in the EP 1 276 586 B1 called.

Advantages of the invention

The laser microdissection system according to the invention has a microscope which has a recording device for receiving a biological sample and a reflected light device with a laser deflection device which guides a laser beam provided by a laser unit through a microscope objective of the microscope onto a sample region of this biological sample and detects the point of impact of the laser beam on the or within the sample area. This laser microdissection system additionally has at least one further laser deflection device, which is set up in such a way that it guides at least one further laser beam onto the mentioned sample area or leads to at least one other sample area and shifts the point of impact of this at least one further laser beam there.

The invention thus employs a further laser beam with associated laser deflection device for manipulating or cutting a sample. Each additional laser beam is assigned a respective laser deflection device. The term "sample area" is intended to mean a region of interest of the specimen under observation, wherein this specimen area is manipulated by means of the incident laser radiation or is cut out by means of a focused laser beam. As a rule, but not necessarily, the laser beam is focused, for example, by a microscope objective on the sample area, in particular for laser cutting.

Due to the use according to the invention of several (at least two) laser beams, three different possibilities of sample processing open up:

First, it is possible to process a single sample area on a sample by a plurality of laser beams, for example to cut a sample area out of the sample by means of a plurality of focused laser beams.

Secondly, two or more sample areas of a microscopically examined biological sample can be processed in parallel and in particular simultaneously with at least one laser beam in each case. For example, multiple sample regions of interest within a sample may be simultaneously dissected out of the sample.

Finally, thirdly, a plurality of sample areas of respectively different samples can be processed in parallel and in particular simultaneously with corresponding laser beams. In this way it is possible, for example, in a series of Sample sections corresponding sample areas in parallel from the respective samples to cut. In the following, this should be referred to as "Parallel Section Cutting" (PSC).

All three of these alternatives can also be combined with each other. For example, the second alternative can be combined with the first alternative, so that several sample areas on a sample are processed in parallel with two or more laser beams in each case. In combinations of the second and third alternative, for example, a plurality of corresponding sample areas on multiple samples would be processed in parallel by means of respective associated laser beams. Those skilled in the art will be aware of the other possible combinations of the first and the third alternative and of all three alternatives.

Before discussing the aforementioned embodiments in more detail, possible and preferred embodiments for the provision of further laser deflection devices with associated laser beams will be discussed below. In general, the further laser beam (or the further laser beams) can be generated by additional laser units and / or by one or more beam splitter arrangements which divide an existing laser beam into two or more beams.

It may be advantageous if the at least one further laser deflection device is assigned a further laser unit which provides one of the at least one laser beam. Thus, at least one further laser deflection device with at least one further laser unit is present. This is particularly useful if the full laser power is required for example for laser cutting for each laser beam.

It may be advantageous if the laser unit is followed by a beam splitter arrangement which generates the at least one further laser beam by beam splitting from the laser beam provided by the laser unit. It would therefore be sufficient in principle if a laser unit is present, from which a plurality of laser beams are generated by beam splitting.

Of course, mixed forms of the two embodiments mentioned may be useful. Thus, a further laser unit can be arranged downstream of a beam splitter arrangement, which in turn generates at least one further laser beam by beam splitting from the further laser beam provided by this further laser unit.

The aforementioned laser deflection devices can guide the respective laser beams in different ways to a sample.

On the one hand, it is possible to guide all the laser beams through the same microscope objective of the microscope of the laser microdissection device. Of course, it is also possible to pass only a part of all available laser beams through the same microscope objective. In this case, the existing laser deflection device and at least one of the (at least one) further laser deflection devices are set up such that the laser beams deflected by these laser deflection devices are guided through the microscope objective of the microscope of the laser microdissection system according to the invention.

On the other hand, a laser focusing lens can be provided for one, several or all of the further laser beams, which guide the respective further laser beam to the same or to another sample. For this purpose, one of the at least one further laser deflection device is set up such that it guides the laser beam deflected by it through a laser focusing lens onto the sample area or onto one of the at least one other sample area. In this embodiment, at least one further laser focusing lens would be added to the microscope or the microscope objective of the laser microdissection system. This is particularly useful if sample areas are to be processed that can not be processed by the same microscope objective, but not another microscope setup is necessary for processing.

Finally, it can also be useful or necessary if the mentioned laser focusing lens represents another microscope objective of another microscope. In this case, the laser microdissection system according to the invention thus comprises several microscopes for the parallel processing of sample areas, as may be of advantage in particular for the above-mentioned parallel section cutting.

Of course, the above-mentioned three embodiments are in turn arbitrarily combined with each other, without the need for further explanation.

For the above-mentioned parallel section cutting (third possibility according to the invention), the laser deflection device of the laser microdissection system according to the invention is set up such that it guides the laser beam assigned to it to the sample area of a first biological sample, wherein at least one further laser deflection device is provided, which is set up such that she leads her each associated further laser beam to each other a different sample area of another sample. This is the parallel operation of multiple laser beams for parallel cutting or manipulating samples possible. For example, several (at least two) laser axes are constructed in parallel with the same optics and directed to sample areas, for example, adjacent serial sections of a preparation. For example, one of the serial sections may be colored so that the sample area of interest can be identified (in a known manner). The other inserted series cuts can be uncoloured. With the aid of the colored section, one or more target areas (sample areas) can be identified and marked. For example, suitable cut lines may be placed around the sample areas of interest to be obtained as dissectates. The markings or cut lines are then correspondingly transferred to the further serial cuts, so that corresponding sample areas on different serial sections can be manipulated or dissected in parallel and collected. The transfer of a mark or cut line is done (in a known manner) via image processing.

Said parallel section cutting, which is intended to include the manipulation of sample areas in addition to the dissection, can be effected by each of the at least one further laser deflection device being assigned a respective laser focusing lens for guiding the respective further laser beam onto the respective sample area. In this case, it would be possible to dispense with a further microscope with a microscope objective for the other laser beams. This is possible in particular if, for example, the serial sections are calibrated perfectly, so that a generated section line in the reference sample can be transferred directly to the parallel serial sections. If, however, further calibration steps are necessary, it is advantageous if the mentioned laser focusing lenses represent further microscope objectives of corresponding microscopes.

For the aforementioned first inventive possibility of parallel cutting or manipulation of a sample area on a sample with at least two laser beams, the laser deflection device of the laser microdissection system according to the invention is set up such that the laser beam is guided onto the sample area of the biological sample and the at least one further laser deflection device is such arranged to guide the at least one further laser beam on the same sample area of this sample. The said sample area can thus be manipulated or cut by a plurality of laser beams. The editing is done in particular on the same field of view as given by the microscope objective. Due to the presence of several laser deflection devices, the laser beams or laser foci can be controlled simultaneously and independently of one another such that a plurality of markings or cutting lines are processed simultaneously. It is also conceivable to work at least partially outside the field of view. Depending on the number of laser beams, a corresponding number of markings or cut lines can be manipulated or cut. It is also possible to cut a cut line with two or more laser foci.

This embodiment leads to a tremendous acceleration of previous laser microdissection methods.

Finally, according to the aforementioned second possibility according to the invention, it is also possible to process a plurality of sample areas in parallel on a sample. For this purpose, the laser microdissection system according to the invention has the laser deflection device, which is set up in such a way that the laser beam is guided onto the sample area of the biological sample, and the at least one further laser deflection device, which is set up such that it projects the at least one further laser beam onto the at least one other Sample area of the same biological sample leads. Concerning embodiments and advantages of this embodiment, reference is made to the previous embodiment just discussed.

In the latter two embodiments (initially mentioned first and second inventive possibilities), it is possible and advantageous to guide the laser beams over the microscope objective of the microscope and to move them by means of the respective laser deflection devices in the respective sample areas. Of course, it may in turn be useful to use one or more other Laserfokussierlinsen next to the existing microscope objective, as already explained above in connection with the Parallel Section Cutting. Mixed forms are also conceivable. In this respect, reference is made to the above explanations.

The invention further relates to uses according to established laser microdissection systems for the parallel cutting or manipulation of a sample area or several sample areas according to the above-described first, second or third inventive option as well as corresponding laser microdissection methods. To avoid repetition, the corresponding uses and laser microdissection methods are shown together below. Further details emerge from the embodiments. For the sake of simplicity, reference shall be made below to the laser microdissection method according to the invention, the statements being read completely analogously to the uses according to the invention.

In the laser microdissection method according to the invention, at least one further laser beam is initially provided. In known manner, a sample area of a biological sample is processed by means of a (first) laser beam. By parallel operation of the at least one further laser beam, at least one further sample area of one or more other samples can be processed in parallel and in particular simultaneously. As already mentioned, "machining" is understood to mean the cutting of a sample area by means of a laser beam or the manipulation of a sample area by means of a laser beam.

The (third) inventive possibility of parallel section cutting has already been discussed above in connection with the laser microdissection system according to the invention. In the present corresponding method, in particular a sample area of a reference sample can be selected as the reference sample area and further sample areas of respectively different samples can be processed by means of respectively assigned laser beams. This type of parallel manipulation or parallel cutting of sample areas is particularly useful if the reference sample area and the other sample areas are components of samples of a serial section of the same preparation. The selection of a reference sample region is carried out in a manner known per se, for example by staining or other contrasting methods. The coordinates of the reference sample area can be transferred to corresponding sample areas of the other samples, wherein, in particular in a serial section, it is to be assumed that the properties of the reference sample area and of the further sample areas determined in this way do not differ or do not differ significantly. The reference sample area is therefore microscopically visualized and marked by means of a microscope or by means of the microscope of the laser microdissection system and then the marking is transferred to the further sample areas. The marking and the transmission of the marking takes place in a manner known per se by means of image processing.

The mentioned method of parallel section cutting is advantageous in particular for the parallel cutting of sample areas corresponding to one another, since the cutting can take place at a significantly higher speed than in the previously known serial section cutting.

A laser microdissection method according to the first possibility of parallel cutting or manipulation according to the invention mentioned above involves the parallel processing of a single sample area by means of a plurality of laser beams. Again, machining by the laser beam may involve cutting the sample area along a predetermined cutting line. Methods for determining suitable cut lines are well known in the art. The inventive method not only allows the parallel cutting by means of several laser beams along such a cutting line, but also in particular the cutting of thick samples, in which case in particular at least one laser beam to the top of the sample area and at least one other laser beam guided to the underside of the sample area or is steered. The laser deflection devices of the corresponding laser beams are set up accordingly for this purpose. For this purpose, corresponding laser focusing lenses can be arranged above and below the sample area to be cut. Alternatively, all laser focusing lenses can be arranged on one side of the sample area, wherein at least one laser beam is deflected to the other side of the sample area by means of deflection devices.

Finally, the laser microdissection method according to the above-mentioned second possibility according to the invention of parallel cutting or manipulation enables the parallel processing of several sample areas of a single sample by means of a plurality of laser beams operated in parallel.

figure description

1 shows a laser microdissection system, which preferably represents the starting point of the present invention, in a schematic representation.

2 shows schematically a possibility according to the invention for the parallel processing of sample areas on different samples.

3 schematically shows a possibility according to the invention of the parallel processing of a sample area on a sample.

4 schematically shows a possibility according to the invention of the parallel processing of one or more sample areas on a sample.

5 schematically shows a possibility according to the invention of the parallel processing of one or more sample areas on a sample.

6 schematically shows a possibility of providing additional laser beams by further laser units.

7 shows schematically the provision of further laser beams by means of a beam splitter arrangement, and

8th schematically shows a combination possibility of the examples of 5 and 6 ,

In the figures, corresponding elements are given identical reference numerals and will not be explained repeatedly.

In 1 For example, a laser microdissection system that can be used to practice the invention is shown schematically and collectively with FIG 100 designated. The laser microdissection system 100 corresponds in essential parts to that in the EP 1 276 586 B1 is disclosed, which is incorporated herein by reference. A coordinate system, by means of which the axes or directions x, y and z mentioned below are illustrated, is shown in FIG 1 With 200 designated.

The laser microdissection system 100 includes a microscope 10 , In a microscope head 11 of the microscope 10 may be a partially illustrated illumination device 12 be provided. This may, 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 diaphragms. For transmitted light illumination and for setting suitable contrast or observation methods, a condenser unit 90 be provided.

The microscope 10 may be designed as a confocal, in particular as a spinning disk microscope and in this case has corresponding further or alternative means (in 1 not shown).

At the microscope head 11 For example, also a user input and / or user information unit 13 arranged, which may be formed, for example, as a touch screen, and via which the user can enter and / or read, for example, viewing and / or editing parameters.

Furthermore, a drive knob 14 intended. This serves to operate a coarse and a fine drive for setting a height of a microscope stage 30 , A sample 51 , For example, one in a corresponding receiving device or holder 52 attached tissue sample, can thereby into an object plane of a lens 41 to be brought. The objective 41 is next to other lenses 42 in a nosepiece 40 attached. To protect against laser radiation, a protective hood 15 be provided.

From the sample 51 outgoing observation light runs along an observation beam path a. In a tube unit 60 with suitable decoupling devices 61 a preferably variable portion of the observation light, for example, by 60 °, and coupled by means of an eyepiece pair 62 presented to a user. Another portion of the observation light may be in a digital imaging unit 63 coupled and recorded by imaging. The image capture unit 63 can, on site, in a control unit 82 or a control computer 81 (see below), or in a different spatial arrangement, an image evaluation module 64 be assigned.

The laser microdissection system 100 has a laser unit 70 with a laser light source 75 on. A through the laser light source 75 , which may be, for example, a UV laser light source, provided laser beam 77 with laser beam axis b is in a Auflichteinheit, here with a total of 76 is indicated at a first deflection mirror 71 and a second deflecting mirror 72 deflected and through the lens 41 to the test 51 in the sample area 50 focused.

In the laser microdissection system 100 may be the location where the laser beam 77 to the test 51 in the object plane, and thus also in the sample area 50 impinges, basically be set in different ways. On the one hand, a manual adjustment 31 be provided by means of which designed as a cross table microscope stage 30 in x- and y-direction (ie here perpendicular or parallel to the paper plane) can be adjusted. In addition to the adjustment 31 can also be provided electromechanical actuating means, for example, by a control unit 82 controlled or their position by the control unit 82 can be detected.

The control unit 82 can also be any other motorized functions of the laser microdissection system 100 control and in particular an interface to an external control computer 81 who has appropriate connections 83 be tethered. The control unit 82 or the control computer 81 can also, for example, by means of the image evaluation module 64 evaluate received data. For example, this may result in a sequence of tissue layers or other structures of the sample 51 be recognized.

For laser microdissection, in particular a laser deflection device can be used 73 be provided. By means of the laser deflection device 73 can the laser beam 77 opposite one between the first deflecting mirror 71 and the second deflecting mirror 72 extending optical axis c are deflected. The laser beam can therefore at different positions on the second deflection mirror 72 impact, which may be formed, for example, as a dichromatic divider, and is therefore also in different positions to the test 51 focused in the object plane. A deflection by means of a laser deflecting device 73 is in the detail in the EP 1 276 586 B1 shown. It should be stressed that here are different ways to deflect a laser beam 77 or for positioning the sample 51 in the object plane opposite to the laser beam 77 can be used. The invention is not limited to the illustrated example.

In the example shown, the laser deflection device 73 two massive glass wedge plates 731 which are inclined against the optical axis c and are rotatable independently of each other about the optical axis c. These are the wedge plates 731 with ball bearings 732 stored. Each of the wedge plates is with a gear 733 connected. The gears 733 can each by means of actuators 734 are rotated, which can be acted upon by corresponding drive signals and correspondingly the gears 733 drive. The rotation devices can via position sensors 735 have (here only on the right actuator 734 shown). One by the posers 735 detected position can be sent to the control unit 82 be transmitted.

2 shows a possibility according to the invention of the parallel processing of sample areas on different samples. Without restricting generality, it will be assumed below that the sample areas are intersected by means of a focused laser beam in order to generate dissected data. This in 2 described embodiment represents an embodiment of the discussed in the description Parallel Section Cutting.

2 shows a lens 41 that has a laser beam 77 on a sample 51 focused. The laser beam 77 is by means of a arranged on the optical axis c laser deflection device 73 in the xy direction within a sample area 510 the sample 51 postponed. For a more detailed explanation of this system, see the Explanatory Notes to the full text 1 directed. For the sake of simplicity, is in 2 the laser microdissection system 100 out 1 not shown in full. Only the components necessary for understanding the invention are shown and described below.

The sample 51 is on a suitable bracket 52 applied, which allows a laser dissection. The sample area, which by means of the focused laser beam 77 is cut out, is with 510 designated. The sample area 510 forms a portion of the sample 51 in which the sample area is identified in a manner known per se as a sample area of interest. This is usually done by microscopic visualization of the sample 51 and the sample area 510 , wherein by coloring the sample 51 and by appropriate (fluorescence) examination of the sample 51 the sample area of interest 510 can be determined. In a manner which is also known per se, this sample area is formed 510 put an example individual cutting line. This process can be done manually or preferably by means of image processing. The result can be, for example, on the external control computer 81 (see. 1 ). The command for cutting the sample is issued 51 , controls the control unit 82 the laser deflector 73 such that the laser beam focus on the sample 51 the section line describes and in this way the sample area 510 separates from the rest of the sample.

In the illustrated Parallel Section Cutting are other samples 53 . 54 taken on appropriate brackets to be processed in parallel. These are other laser beams 77 ' and 77 '' intended. Each of these further laser beams is in each case a further laser deflection device 73 ' . 73 '' assigned. The optical axes are denoted by c 'and c'', respectively. For the samples 53 and 54 are expediently in particular adjacent sections of a serial section of a preparation. Samples 53 and 54 do not necessarily have to be colored. On the contrary, it is beneficial to work with unstained samples, as staining may affect the properties or biological processes of the sample.

The around the sample area 510 the sample 51 fixed cutting line is on the unstained sample areas 530 and 540 transferred so that the sample areas 530 and 540 the reference sample area 510 correspond. The term "transfer of the cutting line" should be understood to mean that control commands are sent to corresponding control units of the laser deflection devices 73 ' . 73 '' be transferred to the laser beam 77 ' respectively. 77 '' according to the specified cutting line on the sample 53 respectively. 54 respectively.

In this way, it is thus possible, according to the once defined cutting line around the reference sample area 510 parallel and in particular simultaneously several corresponding sample areas 530 . 540 from further samples 53 . 54 cut out. Here is the option, even the laser beam 77 deflect accordingly so that its focus is shifted along the cut line to the reference sample area 510 to dissect. However, this need not necessarily be the case if, for example, the colored reference sample area should be less useful or completely useless as a dissectate for further analysis. Because of this, the laser beam is 77 drawn only dashed.

The embodiment according to 2 is of course not limited to the number of samples shown there. As well as already mentioned, other markings can also be used as cutting lines for parallel section cutting. For example, target areas can be identified as sample areas, marked and then manipulated. On the possibilities for the production of further laser beams 77 ' . 77 '' will be discussed in more detail below. In 2 are only the corresponding Laserablenkeinrichtungen 73 ' . 73 '' shown. The laser focusing lenses 41 ' . 41 '' care in 2 for focusing the correspondingly assigned laser beams 77 ' . 77 '' to the respective sample 53 . 54 , If these samples 53 . 54 Precisely calibrated, it may be when using laser focusing lenses 41 ' . 41 '' stay. However, if calibrations are necessary to ensure that corresponding sample areas have the same coordinates, it is advantageous if the illustrated laser focusing lenses 41 ' . 41 '' Microscope lenses of other microscopes (not shown) represent. Such more "microscopes" will be the essential components of the 1 as far as these components enable calibration and subsequent dissection, namely essentially the laser unit, laser deflecting device, lens and recording device for the sample.

3 schematically shows a possibility of processing a sample area in parallel on a sample. Without limiting the generality, it should again be assumed that the sample is cut. As based on 2 already explained above, this is a laser beam 77 on a sample 51 by means of a microscope objective 41 focused. The position of the focus of the laser beam 77 on the test 51 is using a laser deflector 73 controlled.

By means of another laser beam 77 ' with associated further laser deflection device 73 ' can the same sample area 510 the sample 51 to be edited. In particular, both laser beams 77 and 77 ' parallel and in particular at the same time the sample area 510 cut along a cutting line. In this way, the speed of dissecting can be increased. The parallel lasers 77 . 77 ' So you can cut along the same cutting line at different coordinates at the same time, but in principle also at the same coordinates, for example with thick samples. Here are the foci of the laser 77 . 77 ' in different sample depths to cut thick samples faster and more effectively.

By means of corresponding deflection mirrors 72 . 72 ' can the axes b, b 'of the laser beams 77 . 77 ' in the optical axis c of the lens 41 be coupled.

4 schematically shows a possibility according to the invention for the parallel processing of one or more sample areas on a sample. Like reference numerals again designate like elements as in the preceding figures.

The embodiment according to 4 can as a further embodiment of the parallel processing of a sample area 510 on a sample 51 (see. 3 ). In contrast to the embodiment according to 3 are in 4 but two focusing lenses 41 . 41 ' or a microscope objective 41 and a focusing lens 41 ' or two microscope lenses 41 . 41 ' intended. Thus, the two laser beams become 77 . 77 ' not by a single focusing lens or a single microscope objective 41 guided, but by a respective associated microscope objective or an associated focusing lens 41 respectively. 41 ' , This allows, for example, not only within the field of view of a microscope objective, but also outside of this field of view and finally to work within the fields of view of two microscope objectives, depending on whether 41 and 41 ' a focusing lens and a microscope objective or two microscope lenses.

In another embodiment of the example according to 4 hit the laser beams 77 . 77 ' to two different sample areas (not separately indicated). In this way it is possible to have different sample areas of a sample 51 parallel and in particular at the same time to cut or manipulate. Thus, for example, a plurality of labeled sample regions of interest can be simultaneously extracted from a sample 51 be cut, which accelerates the process of laser microdissection enormously. Concerning. For further details of these operations, reference is made to the preceding embodiments.

5 shows a further embodiment of the arrangement according to 4 , Here is an additional third laser beam 77 '' provided in the representation according to 5 on the bottom of the sample 51 is directed while the two laser beams 77 . 77 ' on top of this sample 51 are directed. The on the bottom of the sample 51 directed laser beam 77 '' has an associated laser deflection device 73 '' , In the 5 shown device is in turn suitable for cutting (or other editing) of a single sample area 510 on the test 51 or several sample areas (not separately indicated) on the sample 51 , In this regard, reference should be made to the explanatory notes 4 directed.

The laser beam directed at the underside 77 '' according to 5 can be used in particular for cutting thicker sample areas. For example, the laser beam 77 ' from above on the Cutting line are guided while the laser beam 77 '' from below on the same cutting line, so that the cutting line is cut from two sides. In this way, a thicker sample area can be cut more gently than if a single laser beam of correspondingly high energy were cut from one side only.

Concerning. of the 4 and 5 It should also be emphasized that the oblique position of the laser axes shown there can not necessarily be achieved only by the illustrated oblique position of the optical axes of the microscope objectives or laser focusing lenses. An appropriate angle of incidence of the laser beam on the sample, as shown by the 1 and 3 explained, also and preferably achieved with lenses or Laserfokussierlinsen whose optical axes are perpendicular to the sample, wherein the inclinations of the laser axes are then caused solely by the associated Laserablenkeinrichtungen. The inclinations of the axes in the 4 and 5 thus serve the easier understanding.

6 schematically shows a possibility of providing additional laser beams by further laser units. Here are two laser units 70 and 70 ' in a laser microdissection system 100 available (cf. 1 ). The laser units 70 . 70 ' generate corresponding laser beams 77 . 77 ' , the laser deflection units associated therewith 73 . 73 ' be guided on a sample area and moved there.

7 shows schematically the provision of further laser beams by means of a beam splitter arrangement. The laser unit 70 generates a laser beam, which is a beam splitter arrangement 78 passes. Here, the laser beam is divided into two beams, with one of the two beams 77 is designated. The other of the two divided beams is with 77 ' designated. This allows two laser beams 77 . 77 ' be generated, the intensity of which is correspondingly lower than that of the original laser beam 77 , For example, the laser beam 77 ' on a deflecting mirror 74 reflected and a laser deflecting device associated with it 73 ' be supplied.

While the embodiment according to 6 is particularly useful for cutting processes in which the full laser intensity is needed, the embodiment according to 7 advantageous for processes such as manipulations, for which a portion of the original laser power is sufficient.

Finally shows 8th a combination possibility of the embodiments of the 6 and 7 , This is the one of the laser unit 70 generated laser beam 77 a laser deflector 73 fed. Another laser beam is transmitted through another laser unit 70 ' generated. This further laser beam is split into two laser beams, again with one of the two split laser beams 77 ' the other with 77 '' should be designated. The splitting takes place again in a beam splitter arrangement 78 ' , The partial beams generated in this way 77 ' and 77 '' then each corresponding laser deflection devices 73 ' and 73 '' fed, being for the laser beam 77 '' turn a deflection mirror 74 can be used. An arrangement, as in 8th can be shown, for example, for the parallel cutting of thick samples or sample areas (with laser beam 77 ) and thin samples or sample areas (with the laser beams 77 ' and 77 '' ) be used. The skilled person will be apparent from the 6 to 8th further combination possibilities.

LIST OF REFERENCE NUMBERS

10

microscope

11

the microscope

12

lighting device

13

User input / information unit

14

drive knob

15

guard

30

microscope stage

31

manual adjusting device

40

nosepiece

41

lens

41 ', 41' '

Laser focusing lens, objective

42

lens

51

sample

52

recording device

53

sample

54

sample

510

Sample area, reference sample area

530

sample area

540

sample area

60

barrel unit

61

Auskoppeleinrichtungen

62

eyepieces

63

Image capture unit

64

Image analysis module

70, 70 '

laser unit

71

deflecting

72, 72 '

deflecting

73, 73 ', 73' '

Laser deflector

74

deflecting

75

Laser light source

76

incident-light

77, 77 ', 77' '

laser beam

78, 78 '

Beam splitter arrangement

731

wedge plates

732

ball-bearing

733

gear

734

actuator

735

locator

81

tax calculator

82

control unit

83

links

90

condenser unit

100

laser microdissection system

a

Observation beam path

b

laser beam axis

c

optical axis

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• EP 1276586 B1 [0005, 0022, 0061, 0070]
• US 2012/0045790 A1 [0010, 0011]

Claims (35)

Laser microdissection system ( 100 ) with a microscope ( 10 ) having a receiving device ( 52 ) for receiving a biological sample ( 51 ) and a Auflichteinrichtung ( 76 ) with a laser deflection device ( 73 ), which one through a laser unit ( 70 ) provided laser beam ( 77 ) through a microscope objective ( 41 ) of the microscope ( 10 ) on a sample area ( 510 ) of the biological sample ( 51 ) and which an impact point of the laser beam ( 77 ) on the sample area ( 510 ), characterized in that the laser microdissection system ( 100 ) at least one further laser deflection device ( 73 ' . 73 '' ) for guiding at least one further laser beam ( 77 ' . 77 '' ) on the sample area ( 510 ) or at least one other sample area ( 530 . 540 ) and for displacing the point of impact of the at least one further laser beam ( 77 ' . 77 '' ) on the respective sample area. Laser microdissection system ( 100 ) according to claim 1, characterized in that the at least one further laser deflection device ( 73 ' ) another laser unit ( 70 ' ) associated with one of the at least one laser beam ( 77 ' ). Laser microdissection system ( 100 ) according to claim 1, characterized in that the laser unit ( 70 ) a beam splitter arrangement ( 78 ), which from the laser unit ( 70 ) provided laser beam ( 77 ) the at least one further laser beam ( 77 ' ) generated by beam splitting. Laser microdissection system ( 100 ) according to claim 2, characterized in that the further laser unit ( 70 ' ) a beam splitter arrangement ( 78 ' ), which consists of that of the further laser unit ( 70 ' ) provided further laser beam ( 77 ' ) at least one further laser beam ( 77 '' ) generated by beam splitting. Laser microdissection system ( 100 ) according to one of claims 1 to 4, characterized in that the laser deflection device ( 73 ) and one of the at least one further laser deflection device ( 73 ' ) are arranged such that the laser beams deflected by these laser deflection devices ( 77 . 77 ' ) through the microscope objective ( 41 ) of the microscope ( 10 ). Laser microdissection system ( 100 ) according to one of claims 1 to 4, characterized in that one of the at least one further laser deflection device ( 73 ' ) is set up in such a way that it deflects the laser beam deflected by it ( 77 ' ) by a laser focusing lens ( 41 ' ) on the sample area ( 510 ) or one of the at least one other sample area ( 530 . 540 ) leads. Laser microdissection system ( 100 ) according to claim 6, characterized in that the Laserfokussierlinse another microscope objective ( 41 ' ) represents another microscope. Laser microdissection system ( 100 ) according to one of claims 1 to 7, characterized in that the other sample area another sample area of the same sample ( 51 ) or another sample area ( 530 . 540 ) of another sample ( 53 . 54 ). Laser microdissection system ( 100 ) according to one of the preceding claims, characterized in that the laser microdissection system ( 100 ) the laser deflection device ( 73 ) for guiding the laser beam ( 77 ) on the sample area ( 510 ) a first biological sample ( 51 ) and the at least one further laser deflection device ( 73 ' . 73 '' ) for guiding the at least one further laser beam ( 77 ' . 77 '' ) to one of the at least one other sample area ( 530 . 540 ) of another sample ( 53 . 54 ) or, as far as dependent on claim 8, the other sample ( 53 . 53 ) having. Laser microdissection system ( 100 ) according to one of claims 1 to 8, characterized in that the laser microdissection system ( 100 ) the laser deflection device ( 73 ) for guiding the laser beam ( 77 ) on the sample area ( 510 ) of the biological sample ( 51 ) and the at least one further laser deflection device ( 73 ' . 73 '' ) for guiding the at least one further laser beam ( 77 ' . 77 '' ) on the same sample area ( 510 ) of this biological sample ( 51 ) having. Laser microdissection system ( 100 ) according to one of claims 1 to 8, characterized in that the laser microdissection system ( 100 ) the laser deflection device ( 73 ) for guiding the laser beam ( 77 ) on the sample area ( 510 ) of the biological sample ( 51 ) and the at least one further laser deflection device ( 73 ' . 73 '' ) for guiding the at least one further laser beam ( 77 ' . 77 '' ) on the at least one other sample area of the same biological sample ( 51 ) having. Laser microdissection system ( 100 ) according to claim 9, 10 or 11, characterized in that each of the at least one further laser deflection device ( 73 ' . 73 '' ) each have a laser focusing lens ( 41 ' . 41 '' ) for guiding the respective further laser beam ( 77 ' . 77 '' ) is assigned to the respective sample area. Laser microdissection system ( 100 ) according to claim 12, characterized in that at least one of the Laserfokussierlinsen another microscope objective ( 41 ' . 41 '' ) represents another microscope. Use of a laser microdissection system ( 100 ) according to claim 9 for the parallel processing of sample areas ( 510 . 530 . 540 ) of different samples ( 51 . 53 . 54 ) by means of respectively assigned laser beams ( 77 . 77 ' . 77 '' ). Use according to claim 14, wherein a sample area ( 510 ) a reference sample ( 51 ) is selected as reference sample area and further sample areas ( 530 . 540 ) of different samples ( 53 . 54 ) by means of respectively assigned laser beams ( 77 ' . 77 '' ) to be edited. Use according to claim 15, wherein the reference sample area ( 510 ) and the other sample areas ( 530 . 540 ) Constituents of samples ( 51 . 53 . 54 ) of a serial section of the same preparation. Use according to claim 15 or 16, wherein the reference sample area ( 540 ) by means of the microscope ( 10 ) of the laser microdissection system ( 100 ) is visualized and marked microscopically and the marking on the further sample areas ( 530 . 540 ) is transmitted. Use according to claim 17, wherein the mark represents a cut line representing the reference sample area ( 510 ) surrounds. Use according to claim 18, wherein after transfer of the cutting line the further sample areas ( 530 . 540 ) by means of the associated parallel-operated laser beams ( 77 ' . 77 '' ) from the respective samples ( 53 . 54 ) get cut. Use of a laser microdissection system according to claim 10 for parallel processing of a sample area ( 510 ) of the sample ( 51 ) by means of several laser beams. Use according to claim 20, wherein the machining comprises cutting the sample area ( 510 ) along a cutting line. Use according to claim 21, wherein at least one laser beam ( 77 ' ) on top of the sample area ( 510 ) and at least one other laser beam ( 77 '' ) on the underside of the sample area ( 510 ) to be led. Use of a laser microdissection system according to claim 11 for parallel processing of a plurality of sample areas of a sample ( 51 ) by means of the sample areas of respectively associated laser beams ( 77 ' . 77 '' ).  Use according to claim 23, wherein the machining comprises cutting the sample areas along respective cut lines. Laser microdissection method for processing a sample area ( 510 ) a biological sample ( 51 ) by means of a laser beam ( 77 ), characterized in that at least one further laser beam ( 77 ' . 77 '' ) and at least one further sample area ( 530 . 540 ) of different samples ( 53 . 54 ) as well as optionally the sample area ( 510 ) of the sample ( 51 ) by means of the at least one further laser beam ( 77 ' . 77 '' ) or optionally the laser beam ( 77 ) are processed in parallel. The method of claim 25, wherein a sample area ( 510 ) a reference sample ( 51 ) is selected as reference sample area and further sample areas ( 530 . 540 ) of different samples ( 53 . 54 ) by means of respectively assigned laser beams ( 77 ' . 77 '' ) to be edited. The method of claim 26, wherein the reference sample area ( 510 ) and the other sample areas ( 530 . 540 ) Constituents of samples ( 51 . 53 . 54 ) of a serial section of the same preparation. A method according to claim 26 or 27, wherein the reference sample area ( 510 ) by means of a microscope ( 10 ) is visualized and marked microscopically and the marking on the further sample areas ( 530 . 540 ) is transmitted. The method of claim 28 wherein the marker is a cut line representing the reference sample area ( 510 ) surrounds. The method of claim 29, wherein after transmission of the cut line the further sample areas ( 530 . 540 ) by means of the associated parallel-operated laser beams ( 77 ' . 77 '' ) from the respective samples ( 53 . 54 ) get cut. Laser microdissection method for processing a sample area ( 510 ) a biological sample ( 51 ) by means of a laser beam ( 77 ), characterized in that at least one further laser beam ( 77 ' ) and the sample area ( 510 ) by means of the laser beam ( 77 ) and the at least one further laser beam ( 77 ' ) is processed in parallel. The method of claim 31, wherein the machining comprises cutting the sample area ( 510 ) along a cutting line. Method according to claim 32, wherein at least one laser beam ( 77 ' ) on top of the sample area ( 510 ) and at least one other laser beam ( 77 '' ) on the underside of the sample area ( 510 ) to be led. Laser microdissection method for processing a sample area ( 510 ) a biological sample ( 51 ) by means of a laser beam ( 77 ), characterized in that at least one further laser beam ( 77 ' ) and at least one further sample area of the sample ( 51 ) by means of the at least one further laser beam ( 77 ' ) is processed in parallel.  The method of claim 34, wherein the machining comprises cutting the sample areas along respective cut lines.

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