WO2017211644A1

WO2017211644A1 - Supporting membrane for the laser microdissection of a sample deposited on the supporting membrane, laser microdissection device and method for laser microdissection using such a supporting membrane

Info

Publication number: WO2017211644A1
Application number: PCT/EP2017/063167
Authority: WO
Inventors: Falk Schlaudraff
Original assignee: Leica Microsystems Cms Gmbh
Priority date: 2016-06-10
Filing date: 2017-05-31
Publication date: 2017-12-14
Also published as: DE102016110750A1

Abstract

The present invention relates to a supporting membrane (54) for the deposition thereon of a sample (51), of which at least a part (510) is to be cut out by laser microdissection together with a corresponding part (540) of the supporting membrane (54), wherein the supporting membrane (54) is marked by at least one biologically and/or chemically and/or physically active label (55), wherein at least one corresponding biologically and/or chemically and/or physically active specific bonding partner (56) exists for a specific label (55), which can form a corresponding biological and/or chemical and/or physical bond with the label (55), wherein, for a specific sample (51) to be cut out, at least one label (55) is present on the supporting membrane (54), such that the cut out part (540) of the supporting membrane (54), together with the part (510) of the sample (51), includes this at least one label (55), and wherein for different samples (51, 51') to be cut out, which are deposited on the supporting membrane (54), different labels (55, 55') are present on the supporting membrane (54), wherein for at most one of the specific different samples (51, 51') there is no label present on the supporting membrane (54).

Description

Support membrane for the laser microdissection of a sample applied to the support membrane, laser microdissection device and laser microdissection method using the same

support membrane

description

The present invention relates to a support membrane for applying a sample to be excised at least in part by laser microdissection together with a corresponding part of the support membrane, a use of such a support membrane, a laser microdissection device having such a support membrane, and a laser microdissection method using such a support membrane.

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 collecting container. The dissectate may also be taken from the sample together with a membrane attached to the sample be cut out. 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 described, for example, in EP 1 276 586 B1.

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 juxtaposition of such holes or depressions, optionally with overlap. The laser microdissection can be used for obtaining individual cells or defined tissue areas with diameters of typically less than 250 μηι, but usually only 20 μιτ) or less, which are separated by a laser beam from the surrounding tissue and then subjected to different diagnostic analysis methods, for example. In oncology, for example, laser microdissection can be used to isolate specific (tumor) cells from a microscopic section and to examine them for specific metabolites or proteins.

In the present case, the case of a sample adhered to a support membrane is to be considered, wherein in the laser microdissection at least a portion of this sample is cut out together with a corresponding part of the support membrane.

Different samples or sample parts, which are obtained by means of laser microdissection, must be clearly assigned to the original sample or the source of this sample for subsequent analysis. For this purpose, the dissections of different samples are collected in different collection containers or sample catchers in order to avoid contaminations with foreign samples and to be able to subsequently sort the dissections into different containers for further analysis. So far, there is no way to collect the dissections for different samples in the same solution or in the same buffer one and the same receptacle and after sample microdissection sample specific to separate and supply one or more different analytical methods. Thus, depending on the sample and analysis method, a different collection vessel must be used. This is cumbersome and requires the use of multiple collection vessels during the laser microdissection of a sample, or at least during the laser microdissection of several different samples.

DE 101 35 091 A1 discloses a slide, which is formed from at least two layers of carrier materials, wherein a layer is a carrier film with magnetic properties. A microdissectate can then be taken up into a collecting vessel by means of magnetic attraction from the magnet located below the bottom of the collecting vessel. DE 103 22 348 B4 discloses a device for non-contact transfer of a dissectate into a receptacle, wherein the dissectate electrostatically charged by the cutting process is transferred into the receptacle by means of an electric field generated by an electrode.

According to WO 03/008934 A1, a microdissectate can be selectively displaced by means of a mobilizing means, which in turn is a magnetic or magnetizable connection, via an electromagnetic force as a result of the force interaction. This is intended to solve the prior art problem of inefficient mobilization of the microdissect to release the remainder of the specimen or slide.

DE 198 04 800 C2 discloses a collecting device for membrane-supported micro dissectates, wherein an electrostatic recovery force or a magnetic recovery force is proposed to support the gravitational trapping force.

The present invention therefore has the task of making the process of the laser microdissection simpler, in particular without the disadvantages mentioned, while maintaining the same level of reliability.

Disclosure of the invention

To achieve the stated object, the present invention provides a support membrane, a use of such a support membrane, a laser microdissection device comprising such a support membrane, and finally a laser microdissection method using such a support membrane according to the independent claims. Advantageous embodiments are the subject of the respective subclaims and the following description.

A carrier membrane according to the invention for applying a sample to be excised by means of laser microdissection, at least in part together with a corresponding part of the carrier membrane, is characterized by at least one biologically and / or chemically and / or physically effective label, wherein At least one corresponding, biologically and / or chemically and / or physically effective specific binding partner exists which can form a corresponding biological and / or chemical and / or physical bond with the label. In this way, a clear relationship of sample and label is produced, which later allows an assignment of a microdissectate to the original sample with the help of an inserted binding partner for the label. In order to accomplish this assignment, the label is a biologically and / or chemically and / or physically effective substance (substance). This means that the label can bind to a specific binding partner, which also includes the label being able to interact with such a binding partner that is biologically and / or chemically (also "biochemically") and / or physically conditioned Thus, for example, includes electrostatic, magnetic, chemical or biochemical interactions. Further examples are explained below.

For the present invention, it is not absolutely necessary to mark the entire support membrane with the label, but it is sufficient if at least one label is present on the support membrane for a particular sample to be cut out, that the part cut out together with the part of the sample the carrier membrane contains this at least one label. In this way, several samples with several associated, so sample-specific labein on one and the same support membrane (or, as explained below, also on each different support membrane) are applied. Thus, for certain (or even all) different auszuschneidende samples that are applied to the support membrane, each different label on the support membrane and thus also on the subsequent microdissectat available. Thus, for example, a sample of patient A is assigned a label A 'and a sample of patient B has a label B', in particular on the same carrier membrane. The respectively cut microdissectates contain the label A 'for the piece of tissue of patient A and the label B' for the piece of tissue of patient B. The microdissectates can be collected in the same collecting vessel dry or in a collecting solution (eg PBS), so that no change of a collecting vessel is necessary. Of course, the collecting solution must not have a lysing effect on the microdissectate, since otherwise sample and membrane would be separated from each other with a label. By different or different labels is meant that they are biologically and / or chemically and / or physically effective in different ways and thus can be separated from each other by interaction or binding with a corresponding different corresponding binding partner or a corresponding complementary structure. For example, while the label A 'can form a bond (comprising any specific interaction) with a binding partner A ", the label B' can bind with a binding partner B", so that, for example, in spatially separated locations, in each case one of these binding partners exists, the corresponding labels can be collected and thus be separated from other labels.

For example, for two different samples, it is sufficient if the microdissection of only one sample is provided with a label that specifically binds with a corresponding binding partner (eg at a certain separation site), whereas the microdissectate of the other sample that does not provided with a label, does not undergo such binding and thus can be separated from the microdissectate of the other sample (after it has passed the separation site). This principle also applies to more than two different samples, so that it is sufficient if there is no label on the carrier membrane for (at most) one sample. In other words, for n (with n being a natural number greater than or equal to 2) different samples n-1 or n different specific labels must be present in order to allow a clear separation of the microdissectates.

For clarification, it should be added that not all different samples to be excised must each be labeled with different labels, but that it is possible to make a selection of specific samples which are to be clearly separated later on and which are thus marked with different labels ,

The present invention allows the use of a laser microdissection device with a single sample collector or collecting vessel, through which or different laser microdissectants consisting of a cut-out sample part with carrier membrane part are collected. Since each microdissectat is replaced by a corresponding of the label - with the above-described possible restriction of the number of labels - can then be sorted or separated according to corresponding labels. This allows all microdissectates to be collected in a single receptacle during a laser microdissection. As a result, the method of laser microdissection is significantly simplified and accelerated.

It is furthermore advantageous if the support membrane has the at least one label on a back side, wherein a front side of the support membrane facing away from the rear side is designed for applying the sample. In other words, a sample is applied to the front side of the support membrane and the corresponding label is applied to the back side of this support membrane in the same region of the sample, so that sample and label are cut out together.

The label used is in particular a nucleic acid, a protein, a peptide, a lipid and / or a sugar. The labels may thus be spotted nucleic acids (DNA), for example, which differ in their sequence. The advantage of nucleic acids with a length of only 10 bases lies in the great possibility of combination. For each nucleic acid there is a corresponding specific binding partner (complementary structure). In addition to nucleic acids, proteins, peptides, lipids or sugars, which differ in their surface structures with respect to respective binding partners, are conceivable. In particular, antibodies are suitable as proteins which bind specific binding partners (antigens). From immunology many different antibodies with specific binding partners are known. Binding partners may be, for example, other proteins (including other antibodies), peptides, lipids, or sugars. Of course, antigens can also be used as labels and antibodies for later binding of the specific label. Furthermore, biological molecules that form covalent bonds upon specific contact are good labels and binding partners for later sorting.

Example of nucleic acid (DNA) label:

For example, as a label for a later quadruple separation four different sequences can be used, for example poly-dT for site 1, which later binds specifically to poly-dA; poly-dA for site 2, which later specifically binds to poly-dT; poly- dG for site 3, which later specifically binds to poly-dC; poly-dC for site 4, which later specifically binds to poly-dG (poly = at least 1 base, dA = deoxyribonucleic adenine, dT = deoxyribonucleic thymine, dC = deoxyribonucleic cytosine and dG = deoxyribonucleic guanine). From the molecular biology, the binding properties of the bases are well known: adenine and thymine bind to each other by means of two hydrogen bonds and guanine and cytosine by means of three hydrogen bonds. Combinations of the bases allow a wide spectrum for a wide variety of specific binding sequences.

The current Aufpottungs- or binding process is analogous as for common microarray manufactory and hybridization known (see youtube videos:

https: // www. youtube. com / watch? v = QnqdFFF1 eow

https: // www. youtube. com / watch? v = VNsThMNjKhM).

The use of various specific monoclonal antibodies is well known in the field of immunology. Use examples, as would be useful for the invention proposed here, can be found here:

http://www.retrogenix.com/the4echnoloqv/.

Monoclonal antibodies usually bind specifically a so-called antigen, which is usually a peptide or protein, but also a sugar, lipid or the like. Molecule can be. Also disjointed pairs of specific antibodies and corresponding binding partners can be found here which can be applied to the membrane on the one hand and can be used for sorting the dissectates following the laser microdissection on the other hand.

Possibly. can even design antibodies that specifically bind the currently different LMD carrier membrane (PEN, PET, PPS, POL), so that nothing has to be changed on the membrane, but only following the laser microdissection sorting step using such antibodies for the samples of different membranes can be done. The labels are then inherent to the LMD membrane. This possibility is expressly to be encompassed by the presently claimed invention ("carrier membrane labeled" or "label on carrier membrane present"). In this case, the LMD system (or any other fluorescence microscope) can also be used to verify the specific binding and the autofluorescence of the membrane can be used for identification.

For only two samples to be discriminated, the associated label of one sample may be magnetic, while for the other sample a non-magnetic label or no label (if the carrier membrane is non-magnetic) is used. It is assumed that the sample itself is not magnetic. By "magnetic" should also fall magnetizable. For example, magnetic labels would be magnetic metal coated membranes, while nonmagnetic ones would be those without such a magnetic metal. The same applies to a hydrophilic label, the other sample then having a hydrophobic label (or no label if the support membrane and the microdissectate are hydrophobic). Because of its magnetic or hydrophilic nature, such a label can be clearly distinguished and separated from another label (or only the microdissectate support membrane) with a non-magnetic or hydrophobic property.

Conveniently, the label is spotted or printed (e.g., via bioprinting) on the support membrane by a chemical or biochemical reaction, that is, physically and / or chemically and / or biologically bonded to the support membrane.

The invention further relates to the use of an above-described carrier membrane for the laser microdissection of a sample applied to the carrier membrane. Reference is made to the above explanations.

The invention further relates to a laser microdissection device in which the sample to be dissected is applied to a carrier membrane according to the invention. Also in this regard, reference is made to the above statements.

A laser microdissection device according to the invention is based on a laser microdissection system known per se with a microscope, which has a laser light source for generating a laser beam, a deflection device for deflecting the laser beam, an incident light device for focusing the laser beam through a microlaser. Roskopobjektiv on a sample, a microscope stage with a slide with a dissected on the support membrane applied to dissecting sample and finally a sample side of the slide arranged Lasermikrodissektat-Probenfänger encompassed.

A corresponding laser microdissection system is explained in detail below with reference to FIG. 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 on 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 that have already been prepared for microscopy. These may be, for example, tissue thin sections which have been separated out of a larger tissue block by means of a microtome. 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. A microtome distinguishes Among other things, his company is fundamentally influenced by a laser micro dissection system, which produces cuts with as homogeneous a cutting force as possible. Microtomes are therefore designed to produce a large number of identical cuts with parallel cut surfaces, whereas laser microdissection systems are set up for separating 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 parts, 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.

Finally, the present invention relates to a laser microdissection method for cutting at least a portion of a first sample deposited on a first support membrane together with a corresponding portion of the first support membrane, wherein at least one first biologically and / or chemically and / or physically effective label is such is present on or applied to the first support membrane, that part of the first support membrane cut out together with the part of the first sample contains this first label, and wherein the cut-out part of the first sample together with the corresponding part of the first support membrane of a laser microdissectate At least a first, corresponding, biologically and / or chemically and / or physically effective specific binding partner exists with the first label, a biological and / or chemical and / or ph can enter ysikalische bond.

In the method according to the invention, in addition to the first sample, at least a portion of a second sample, which is different from the first sample, is cut out simultaneously or with a time offset. For this purpose, the second sample is applied to the first or to a second carrier membrane. For this second sample either no label on the support membrane is present (see the above comments on the possible limitation of the number of labels) or at least a second label such on this first support membrane or on this second support membrane or is applied there that together with the part of the second sample cut out part the carrier membrane contains this second label. Again, there is a second, corresponding specific binding partner to the second label. The second label is different to the first label. In addition, the microdissectate, which contains the second label, as possible no components or specific binding properties of the first label included in order to perform a later sorting and / or separation optimally.

For further details and advantages of this method, reference is made to the above statements.

Of course, in principle - as before - the first sample can be recorded in a first sample catcher and the second sample in a second sample catcher. The actual advantage of the invention, however, is that the cut-out parts of the first sample and the second sample are taken up together by a single laser microsiectatic sample catcher. Thus, no change of the sample catcher (collection vessels) is necessary, so that the laser microdissection can be performed with little effort.

In this case, the cut-out portions of the first and second samples may be separated from one another by being supplied to at least two spatially separated separation sites, wherein a first separation site comprises the at least one first specific binding partner corresponding to the first label, and a second separation site Separation point having at least a second, corresponding to the second label specific binding partner. The microdissectate labeled with the first label can in this way interact with the corresponding first binding partner and in this connection enter into a binding according to the key-lock principle. The same applies to the second label and its corresponding second binding partner. Since the two binding partners interact in spatially separated locations, a separation of the microdissectates can take place in this way. A separation can, as already explained in detail above, also take place if (at most) one sample does not carry a label, since then all label-labeled sample microdissectates remain at the associated separation sites, while the unlabeled microdissectates are transported separately from the others , Preferably, after separating the cut-out portions of the first and second samples, that is, after separating the microdissectates, these portions are separately supplied to an analyzing device.

It is particularly advantageous if the first and the second sample are applied to a front side of the first carrier membrane. On a rear side of the first carrier membrane facing away from the front side, in particular, a first label is applied in such a way or is applied in such a way that the part of the first carrier membrane cut out together with the part of the first sample contains this first label. Furthermore, in particular a second label (if necessary) is applied to the back of the first support membrane or applied in such a way that the part of the first support membrane cut out together with the part of the second sample contains this second label. In this way, by the manner of separation described above, the microdissectate of the first sample can be easily separated from the microdissectate of the second sample.

Of course, two support membranes may also be used, with the first sample applied to a front side of the first support membrane and the second sample applied to the front side of the second support membrane. It is particularly advantageous if the first label is applied to a rear side of the first carrier membrane facing away from the front side, and the second label (if necessary) is applied or applied (if necessary) on a rear side of the second carrier membrane facing away from the front side.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination indicated, but also in other combinations or in isolation, without departing from the scope of the present invention.

The invention is illustrated schematically with reference to an embodiment in the drawing and will be described in detail below with reference to the drawing. figure description

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

FIG. 2 schematically shows a situation in laser microdissection in one

Lasermikrodissektionsverfahren.

Figure 3 shows schematically the situation after completion of a laser microdissection with microdissect produced.

Figures 4 and 5 show the analogous representations of Figures 2 and 3 with another sample.

FIG. 6 shows a sampler with various microdissectates.

FIG. 7 shows the microdissectates from FIG. 6 with their labels on the way to the corresponding binding partners.

Figure 8 shows the samples with their labein bound to the corresponding specific binding partner.

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

In FIG. 1, a laser microdissection system or an abovementioned laser microdissection device, which can be used to carry out the invention, is shown schematically and designated by 100 as a whole. The laser microdissection system 100 corresponds in substantial parts to that disclosed in EP 1 276 586 B1, to which reference is expressly made. A coordinate system, by means of which the axes or directions x, y and z mentioned below are illustrated, is denoted by 200 in FIG. The laser microdissection system 100 comprises a microscope 10. In a microscope head 1 1 of the microscope 10, an illumination device 12, which is only partially illustrated here, can 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 can 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 (not shown in FIG. 1).

At the microscope head 1 1, for example, a user input and / or user information unit 13 may be arranged, which may be formed, for example, as a touch screen, and via which the user can input and / or read, for example, viewing and / or processing parameters.

Furthermore, a drive knob 14 is provided. This serves to operate a coarse and a fine drive for adjusting a height of a microscope stage 30. A sample 51, for example a tissue sample attached in a corresponding slide or holder 52, can thereby be brought into an object plane of an objective 41. The objective 41 is fastened next to other objectives 42 in a nosepiece 40. To protect against laser radiation, a protective cover 15 may be provided.

Observation light emanating from the sample 51 runs along an observation beam path a. In a tube unit 60 with suitable coupling-out devices 61, a preferably variable portion of the observation light, for example by 60 °, can be coupled out and presented to a user by means of an eyepiece pair 62. Another portion of the observation light can be coupled into a digital image acquisition unit 63 and detected by imaging. The image acquisition unit 63 can, on site, in a control unit 82 or a control computer 81 (see below), or in a different spatial arrangement, be associated with an image evaluation module 64. The necessary connections to the control computer are designated 83.

The laser microdissection system 100 has a laser unit 70 with a laser light source 75. A provided by the laser light source 75, which may be, for example, a UV laser light source, laser beam 77 with laser beam axis b is deflected in a Auflichteinrichtung, which is indicated here at 76 as a whole, at a first deflecting mirror 71 and a second deflecting mirror 72 and focused by the lens 41 on the sample 51.

In the laser microdissection system 100, the location at which the laser beam 77 impinges on the sample 51 in the object plane, and thus also in the sample area, can basically be set in different ways. On the one hand, a manual adjusting device 31 can be provided, by means of which the microscope stage 30 designed as a cross table can be adjusted in the x and y directions (that is to say, perpendicular to or parallel to the plane of the paper). In addition to the adjusting device 31, it is also possible to provide electromechanical actuating means which can be actuated, for example, by a control unit 82 or whose position can be detected by the control unit 82.

The control unit 82 may 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 may be connected via corresponding connections 83. The control unit 82 or the control computer 81 can also evaluate data obtained, for example, by the image evaluation module 64. By way of example, a sequence of tissue layers or other structures of the sample 51 can thereby be recognized.

For laser microdissection, in particular a laser deflection device 73 can be provided. By means of the laser deflection device 73, the laser beam 77 can be deflected relative to an optical axis c extending between the first deflection mirror 71 and the second deflection mirror 72. The laser beam can therefore impinge on the second deflection mirror 72 at different positions, which can be embodied, for example, as a dichromatic divider, and thus also becomes active different positions focused on the sample 51 in the object level. A 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 possibilities for deflecting a laser beam 77 or for positioning the sample 51 in the object plane relative to the laser beam 77 can be used here. The invention is not limited to the illustrated example.

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 are rotatable independently of each other about the optical axis c. For this purpose, the wedge plates 731 are mounted with ball bearings 732. Each of the wedge plates is connected to a gear 733. The gears 733 can each be rotated by means of actuators 734, which can be acted upon by corresponding drive signals and accordingly drive the gears 733. The rotators may have position sensors 735 (shown here only on the right actuator 734). A position detected by the position sensors 735 may be transmitted to the control unit 82.

With the laser microdissection device 100 described here, at least part of a sample 51 located on the object carrier 52 can be cut out by means of a laser microdissection method. This is explained by way of example as follows with reference to FIGS. 2 and 3:

In a manner known per se, for example, the sample 51 is visualized or the corresponding image of the sample 51 is fed to an image processing system for automatic processing. A sample region of interest may be made visible for example by staining or made usable for image processing. This sample area should be made available as a dissectate for further analysis. For this purpose, the sample area is surrounded, for example, with an individual cutting line, this cutting line imaging the object contour of the sample area. In practice, the cutting line will be spaced from the actual sample area by a certain Δ to not destroy the sample area in the edge area due to the finite diameter of the laser focus during cutting. In a manner known per se, the laser beam 77 is displaced by means of the deflection device 73 according to the predetermined individual cutting line by means of the laser microdissection device 100 shown in FIG. 1, so that the laser beam focus describes the cutting line. For this purpose, the laser deflection device 73 is controlled accordingly by means of the control unit 82 (compare the above explanations). After describing the line of intersection through the laser beam focus, the cut microdissect falls by gravity into a laser microdissect sampler 53 (not shown in FIG. 1).

FIG. 2 very schematically shows the situation at the beginning of a laser microdissection process. Shown are only the essential components of the laser microdissection device 100 from FIG. 1, namely the microscope objective 41 used for focusing the laser beam 77 and the focused laser beam 77, the focus of which is directed essentially onto the sample 51. The slide is indicated at 52. The laser microdissects sampler is located below sample 51 and designated 53. This sampler 53 is also shown very schematically in order to clarify the basic principles of the present invention. The sample 51 is located on a support membrane 54, which in turn is held by the slide or the holder 52. In this exemplary embodiment, the support membrane 54 as a whole, or at least in the region of the microdissection to be produced, contains a biologically and / or chemically and / or physically effective label 55.

FIG. 3 shows the situation immediately after the laser beam focus has described the cutting line explained above to cut out a part 510 of the sample 51. In this way, a corresponding part 540 of the support membrane 54, which is firmly connected to the sample 51, cut out. The microdissect produced in this way, consisting of part 540 of the support membrane 54 and part 510 of the sample 51, falls gravitationally into the sample catcher 53.

As support membrane 54, the known materials PEN, PET, POL or PPS come into question. The sampler 53 may already contain a liquid or be dry. In the case of a liquid, this must not have a lysing effect on the microdissectate, otherwise sample part 510 and carrier membrane part 540 would be separated with label 55. FIGS. 4 and 5 show analogous situations to FIGS. 2 and 3, wherein a second sample 51 'is to be laser microdissected, which is located on a second carrier membrane 54'. The sampler is again denoted by 53. In particular, this is the same sampler used in FIGS. 2 and 3. Since the situations in FIGS. 4 and 5 are completely analogous to those in FIGS. 2 and 3 already discussed, further explanation is omitted in order to avoid repetition. The sample part 510 'cut out of the second sample 51', together with the corresponding cut-out carrier membrane part 540 ', falls into the sample catcher 53, the membrane part 540' having a second label 55 '. Advantageously, the microdissectates with the sample part 510 and the sample part 510 'are collected in the same sampler 53.

FIG. 6 shows the sample catcher 53 after laser microdissection of a number of (in this case four) samples (indicated by different hatching), with several dissectates being cut out for each sample. Dissectates for a given sample are labeled with the same label. The dissectates to different samples are labeled with different labels. The different labels are identified in Figure 6 by different hatching. In Figure 6, the various microdissectates are solubilized and then transferred to a prepared support 57 with spiked corresponding binding partners.

The corresponding specific binding partners to the labels 55, 55 ', 55 ", 55"' are denoted by 56, 56 ', 56 ", 56"' in FIG. After a short incubation, the microdissectates will find their corresponding specific binding partners 56, 56 ', 56 ", 56"' by means of the support membrane labels 55, 55 ', 55 ", 55" and thus sorted on the support 57, as shown in FIG , The microdissects sorted in this way can now be supplied to follow-up analyzes, whereby it is known which sample is at which position of the support 57, which contributes to a suitable selection of the analysis method or objective of the analysis. The procedure proposed here requires significantly fewer sample preparation and separation steps as well as fewer collecting containers; in particular, it comes with a single sampler 53. For example, the membrane areas with the associated sample sections 55, 55 ', 55 "and 55"' (see Figure 6) are different in antigen and the antibody 56 (specifically binding only 55) 56 '(which specifically binds only) 55 '), 56 "(which specifically binds only 55") and 56 "' (which specifically binds only 55"') are spotted on support 57 (see Figure 7), then only the mixture of the dissectates of Figure 6 has to be spotted in solution (for example PBS) and applied to the support 57 and incubated. Due to the key-lock principle, the antigens bind to the appropriate antibodies and thus the separation and sorting process is accomplished.

The method of using specific antibodies or antigens is well known from ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay). For example, murine monoclonal antibodies can be used as "antigens" for rabbit or horse anti-mouse antibodies, unlike ELISA or EIA, specific binding of antigen and antibody is sufficient here. To design specific antibodies for the various plastic types of the LMD membranes to ensure specific binding after laser microdissection in the separation step, the labels are then inherent in the LMD membrane.

The spotting of antibodies or proteins, as well as the use of antibody arrays is known, e.g. visible here:

httpsi // www.youtube.com / watch? v = -CM5rABib60

https://www.youtube.com/watch?v=o1 BDvYEfTFA

Instead of the laser microdissection sample catcher 53 explained here with transfer into the planar support 57, it is also possible with advantage to use capillary sample catchers, as are known from the applicant's patent application DE 10 2013 209 455.8. There, the different capillary chambers and / or different collection spots, for example, at branch lines containing the corresponding binding partner of the membrane label to achieve a separation of the different sample parts. With regard to the construction of such a capillary sampler, reference is expressly made to the said German patent application. Another alternative possibility of separating the different membrane labels by the corresponding binding partners is the so-called Microfluidics Device from Fluidigm. The various carrier membrane parts with the various labein can be distributed by such a Microfluidicaufbau on different chambers, wherein the collecting spots respectively corresponding binding partners are included. For further details of such microfluidics devices, reference is made to the state of the art published by Fluidigm in this regard.

LIST OF REFERENCE NUMBERS

10 microscope

1 1 microscope head

12 lighting device

13 User input / information unit

14 drive button

15 protective cover

30 microscope stage

31 manual adjusting device

40 nosepiece

1 lens

42 objective 1, 51 'sample

2 slides, holder

3 sample catchers

4, 54 'carrier membrane

5, 55 ', 55 ", 55'" label

6, 56 ', 56 ", 56"' binding partner

7 carriers

10, 510 'part of the sample 540, 540 'part of the support membrane

60 tube unit

61 decoupling devices

62 eyepiece pair

63 image capture unit

64 image evaluation module

70 laser unit

71 deflecting mirror

72 deflection mirror

73 laser deflecting device

75 laser light source

76 incident light device

77 laser beam

731 wedge plates

732 ball bearings

733 gear

734 actuator

735 position transmitter

81 control computer

82 control unit

83 connections

90 condenser unit

100 laser microdissection system, device

200 coordinate system

a observation beam axis b laser beam axis

c optical axis

Claims

Patent claims

1. Carrier membrane (54) for applying a sample (51) to be cut out at least in part (510) together with a corresponding part (540) of the carrier membrane (54) by means of laser microdissection, the carrier membrane (54) being cut out by means of at least one biological and/or chemically and/or physically active label (55), whereby for a specific label (55) there is at least one corresponding, biologically and/or chemically and/or physically active specific binding partner (56), which is linked to the label (55). corresponding biological and/or chemical and/or physical bond can form, whereby

For a specific sample (51) to be cut out, at least one label (55) is present on the carrier membrane (54) in such a way that the part (540) of the carrier membrane (54) cut out together with the part (510) of the sample (51) has this at least contains a label (55) and where

For certain different samples (51, 51') to be cut out which are applied to the carrier membrane (54), different labels (55, 55') are present on the carrier membrane (54), with at most one of the certain different samples (51 , 51 ') there is no label on the carrier membrane (54).

2. Carrier membrane (54) according to claim 1, characterized in that the carrier membrane (54) has the at least one label (55) on a back, a front side of the carrier membrane (54) facing away from the back being designed for applying the sample (51). is.

3. Carrier membrane (54) according to one of the preceding claims, characterized in that the at least one label (55) is a nucleic acid, a protein, a peptide, a lipid and / or a sugar.

4. Carrier membrane (54) according to one of claims 1 to 2, characterized in that the at least one label (55) has a magnetic or non-magnetic component and / or a hydrophilic or hydrophobic component.

5. Carrier membrane (54) according to one of the preceding claims, characterized in that the label (55) is spotted onto the carrier membrane.

6. Use of a carrier membrane (54) according to one of claims 1 to 5 for the laser microdissection of a sample (51) applied to the carrier membrane (54).

7. Laser microdissection device (100), which has a laser light source (75) for generating a laser beam (77), a deflection device (73) for deflecting the laser beam (77), an incident light device (76) for focusing the laser beam (77) through a microscope objective ( 41) on a sample (51), a microscope table (30) with a microscope slide (52) with a sample to be dissected (51) applied to a carrier membrane (54) and a laser microdissectate sample catcher (53) arranged on the sample side of the microscope slide (52) comprises, wherein the carrier membrane (54) is designed according to one of claims 1 to 5.

8. Laser microdissection method for cutting out at least a part (510) of a first sample (51) which is applied to a first carrier membrane (54), together with a corresponding part (540) of the first carrier membrane (54), wherein at least a first, biological and/or chemically and/or physically active label (55) is present or applied to the first carrier membrane (54) in such a way that the part (540) of the first carrier membrane cut out together with the part (510) of the first sample (51). (54) contains this first label (55), and wherein the cut out part (510) of the first sample (51) is picked up together with the corresponding part (540) of the first carrier membrane (54) by a laser microdissect sample catcher (53), wherein at least one first, corresponding, biologically and/or chemically and/or physically active specific binding partner exists for the first label (55), which can form a biological and/or chemical and/or physical bond with the first label (55), wherein in addition to the first sample (51), at the same time or with a time offset, at least a part (510 ') of a second sample (51 ') different from the first, which is applied to the first (54) or a second (54') carrier membrane, is cut out, with either no label being present on the carrier membrane for this second sample (51 '). which is or at least a second, biologically and/or chemically and/or physically active label (55') which is different from the first label (55) is present on this first carrier membrane (54) or this second carrier membrane (54') or is applied that the part (540, 540') of the carrier membrane (54, 54') cut out together with the part (510 ') of the second sample (51 ') contains this second label (55'), being to the second label (55') at least one second, corresponding, biologically and/or chemically and/or physically active specific binding partner (56') exists, which can enter into a biological and/or chemical and/or physical bond with the second label (55'). , and wherein the cut out parts (510, 510') of the first and second samples are picked up together by the one laser microdissect sample catcher (53).

9. Laser microdissection method according to claim 8, wherein the cut parts (510, 510 ') of the first and second samples are separated from one another by being fed to a first separation point, the first separation point containing the at least one first label ( 55) has corresponding specific binding partner (56).

10. Laser microdissection method according to claim 9, wherein the cut parts (510, 510 ') of the first and second samples are separated from each other by additionally feeding them to a second separation point spatially separated from the first separation point, the second separation point being at least has a second specific binding partner (56') corresponding to the second label (55').

1 1. Laser microdissection method according to claim 9 or 10, wherein after separating the cut out parts (510, 510 ') of the first and second samples, these parts are fed separately from one another to an analysis device.

12. Laser microdissection method according to one of claims 8 to 1 1, wherein the first and second samples (51, 51 ') are applied to a front side of the first carrier membrane (54).

13. Laser microdissection method according to claim 12, wherein on a back side of the first carrier membrane (54) facing away from the front, a first label (55) is or is applied to the first carrier membrane (54) in such a way that it is together with the part (510) of the first Sample (51) cut out part (540) of the first carrier membrane (54) contains this first label (55).

14. Laser microdissection method according to claim 12 or 13, wherein a second label (55 ') is or is applied to the back of the first carrier membrane (54) in such a way that it is cut out together with the part (510') of the second sample (51 '). Part (540) of the first carrier membrane (54) contains this second label (55 ').

15. Laser microdissection method according to one of claims 8 to 1 1, wherein the first sample (51) is applied to a front side of the first carrier membrane (54) and the second sample (51 ') is applied to a front side of the second carrier membrane (54').

16. Laser microdissection method according to claim 15, wherein the first label (55) is or is applied to a back side of the first carrier membrane (54) facing away from the front side.

17. Laser microdissection method according to claim 15 or 16, wherein the second label (55') is or is applied to a back side of the second carrier membrane (54 ') facing away from the front side.