WO2017211644A1 - Membrane de support pour la microdissection laser d'un échantillon appliqué sur la membrane de support, dispositif de microdissection laser et procédé de microdissection laser utilisant une telle membrane de support - Google Patents

Membrane de support pour la microdissection laser d'un échantillon appliqué sur la membrane de support, dispositif de microdissection laser et procédé de microdissection laser utilisant une telle membrane de support Download PDF

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Publication number
WO2017211644A1
WO2017211644A1 PCT/EP2017/063167 EP2017063167W WO2017211644A1 WO 2017211644 A1 WO2017211644 A1 WO 2017211644A1 EP 2017063167 W EP2017063167 W EP 2017063167W WO 2017211644 A1 WO2017211644 A1 WO 2017211644A1
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Prior art keywords
label
sample
carrier membrane
laser
laser microdissection
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PCT/EP2017/063167
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German (de)
English (en)
Inventor
Falk Schlaudraff
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Leica Microsystems Cms Gmbh
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Publication of WO2017211644A1 publication Critical patent/WO2017211644A1/fr

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2833—Collecting samples on a sticky, tacky, adhesive surface
    • G01N2001/284—Collecting samples on a sticky, tacky, adhesive surface using local activation of adhesive, i.e. Laser Capture Microdissection

Definitions

  • 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.
  • 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.
  • laser capture microdissection 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 Dissektatauffang practicers.
  • upwardly transported dissectates may also be attached to the bottom of a dissectate collection container provided with an adhesive coating.
  • 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.
  • 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 ⁇ , but usually only 20 ⁇ ) 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the label is a biologically and / or chemically and / or physically effective substance (substance).
  • 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
  • a binding partner that is biologically and / or chemically (also “biochemically") and / or physically conditioned
  • biologically and / or chemically also “biochemically”
  • biochemically also “biochemically”
  • physically conditioned includes electrostatic, magnetic, chemical or biochemical interactions. Further examples are explained below.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DNA nucleic acids
  • proteins, peptides, lipids or sugars which differ in their surface structures with respect to respective binding partners, are conceivable.
  • 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.
  • antigens can also be used as labels and antibodies for later binding of the specific label.
  • biological molecules that form covalent bonds upon specific contact are good labels and binding partners for later sorting.
  • 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
  • dG deoxyribonucleic guanine
  • 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.
  • 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.
  • antigen usually a peptide or protein, but also a sugar, lipid or the like.
  • Molecule can be.
  • 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.
  • 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.
  • 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 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. Roskop Acceptiv 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-Probenfnatureer encompassed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a second sample 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.
  • the second sample is applied to the first or to a second carrier membrane.
  • 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.
  • there is a second, corresponding specific binding partner to the second label is different to the first label.
  • 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.
  • 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 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.
  • no change of the sample catcher (collection vessels) is necessary, so that the laser microdissection can be performed with little effort.
  • 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.
  • 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.
  • first and the second sample are applied to a front side of the first carrier membrane.
  • 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.
  • a second label 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.
  • first 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.
  • 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
  • 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.
  • 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.
  • the laser microdissection system 100 comprises a 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.
  • a condenser unit 90 can be provided for transmitted light illumination and for setting suitable contrast or observation methods.
  • 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).
  • 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.
  • 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.
  • a protective cover 15 may be provided.
  • Observation light emanating from the sample 51 runs along an observation beam path a.
  • 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 Auflicht driving, 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.
  • 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.
  • 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).
  • 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.
  • a laser deflection device 73 can be provided.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sample area is surrounded, for example, with an individual cutting line, this cutting line imaging the object contour of the sample area.
  • the cutting line will be spaced from the actual sample area by a certain ⁇ to not destroy the sample area in the edge area due to the finite diameter of the laser focus during cutting.
  • 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.
  • the laser deflection device 73 is controlled accordingly by means of the control unit 82 (compare the above explanations).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the membrane areas with the associated sample sections 55, 55 ', 55 "and 55"' 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.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • 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.
  • EIA enzyme immunoassay
  • capillary sample catchers 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.
  • 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.
  • 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 Microfluidic equal on different chambers, wherein the collecting spots respectively corresponding binding partners are included.

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  • Sampling And Sample Adjustment (AREA)

Abstract

La présente invention concerne une membrane de support (54) pour l'application d'un échantillon (51) à découper par microdissection laser au moins en partie (510) conjointement avec une partie (540) correspondante de la membrane de support (54), la membrane de support (54) étant identifiée au moyen d'au moins une étiquette (55) agissant biologiquement et/ou chimiquement et/ou physiquement, au moins un partenaire de liaison (56) correspondant spécifique à une étiquette (55) déterminée et agissant biologiquement et/ou chimiquement et/ou physiquement existant, lequel partenaire de liaison peut former une liaison biologique et/ou chimique et/ou physique correspondante avec l'étiquette (55), au moins une étiquette (55) étant présente sur la membrane de support (54) pour un échantillon (51) déterminé à découper, de telle sorte que la partie (540) de la membrane de support (54) découpée conjointement avec la partie (510) de l'échantillon (51) contient cette au moins une étiquette (55), et différentes étiquettes (55, 55') étant respectivement présentes sur la membrane de support (54) pour des échantillons (51, 51') déterminés différents à découper qui sont appliqués sur la membrane de support (54), aucune étiquette n'étant présente sur la membrane de support (54) pour au plus l'un des échantillons (51, 51') déterminés différents.
PCT/EP2017/063167 2016-06-10 2017-05-31 Membrane de support pour la microdissection laser d'un échantillon appliqué sur la membrane de support, dispositif de microdissection laser et procédé de microdissection laser utilisant une telle membrane de support WO2017211644A1 (fr)

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DE102016110750.6 2016-06-10
DE102016110750.6A DE102016110750A1 (de) 2016-06-10 2016-06-10 Trägermembran für die Lasermikrodissektion einer auf die Trägermembran aufgebrachten Probe, Lasermikrodissektionseinrichtung und Lasermikrodissektionsverfahren unter Verwendung einer solchen Trägermembran

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