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WO2010034289A1 - Procédé de transport induit par laser de matières depuis un substrat de support transparent - Google Patents

Procédé de transport induit par laser de matières depuis un substrat de support transparent Download PDF

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Publication number
WO2010034289A1
WO2010034289A1 PCT/DE2009/001297 DE2009001297W WO2010034289A1 WO 2010034289 A1 WO2010034289 A1 WO 2010034289A1 DE 2009001297 W DE2009001297 W DE 2009001297W WO 2010034289 A1 WO2010034289 A1 WO 2010034289A1
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WO
WIPO (PCT)
Prior art keywords
substrate
laser
sample
laser light
focus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/DE2009/001297
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German (de)
English (en)
Inventor
Alfred Vogel
Andreas Gebert
Maike Blessenhol
Sebastian Eckert
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Universitaet zu Luebeck
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Universitaet zu Luebeck
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Filing date
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Publication of WO2010034289A1 publication Critical patent/WO2010034289A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G01N2001/2886—Laser cutting, e.g. tissue catapult

Definitions

  • the invention relates to a method for the laser-induced transport of materials, in particular of biological materials such as single cells or tissue samples, of transparent carrier substrates in collecting devices.
  • the processing with mechanical manipulators always involves the risk of contamination of the sample by substances with which the manipulator was previously or temporarily in contact and is also time-consuming and hardly automatable.
  • For the removal of a large number of samples from a biological mass is the computer-aided selection with accompanying laser-induced transport of the sample material prior art.
  • the sample is scanned with a focused, pulsed laser beam and the selected areas are ejected by applying a sufficiently large pulse energy (“catapulted"), with a collecting device (eg the lid of the analysis vessel or a microtiter plate) arranged in the foreseeable trajectory of the sample is.
  • a collecting device eg the lid of the analysis vessel or a microtiter plate
  • the biological mass is located on a transparent substrate (for example a glass slide) and is irradiated through this substrate with a laser
  • a transparent substrate for example a glass slide
  • a laser Usually a UV laser, whose radiation is well absorbed by biological materials, is cut out of the mass by laser irradiation and then directly heated and partially vaporized by a "transport pulse” directed at the bottom of the sample.
  • the laser-induced steam expansion serves as the driving force of the transport.
  • catapulting directly from glass is detrimental to the biological material (Vogel A et al.
  • thermomechanical damage occurs in the irradiated area and the UV radiation in high dose on the material.
  • the yield of recoverable genetic material for Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis is lower by a factor of about 100-1000 than with methods that provide support for catapulting through additional layers between substrate and sample ,
  • DE 196 03 996 C2 discloses a method for separating and sorting biological objects which are arranged on a planax carrier, wherein an object field of the carrier, on which a selected biological object is located, is cut out with a laser beam and then transported laser-induced.
  • the carrier is so as
  • the biological sample is supported by the mechanical stability of the carrier before and during transport.
  • the laser pulse which triggers the transport, is directed onto the carrier, which releases the energy required for accelerating the dissectate by local evaporation.
  • the biological sample itself which is located inside a closed laser cut line, which can enclose an area much larger than the laser spot, is not affected in all areas outside the laser focus by the UV radiation and the heat development in the laser focus.
  • the carrier from DE 196 03 996 C2 is commercially available today and consists of a thin film of polyethylene-2,6-naphthalate (PEN).
  • PEN polyethylene-2,6-naphthalate
  • the PEN film is only fixed at its edges on the slide, whereby the samples lie loose after cutting out the relevant areas of the tissue and are very easy to catapult.
  • the lack of adhesion of the film and its high absorbance in the laser wavelength range allow catapulting the dissectate even when only a fraction of the cut surface is illuminated.
  • the use of the PEN film is not without its disadvantages.
  • the optical imaging performance is reduced (and thus, for example, the visibility of histological details). Scattering and birefringence limit the possibility of optical contrasting by phase or interference contrast.
  • the film has considerable autofluorescence in the blue and green spectral range, so that green fluorescent Marks to detect relevant tissue areas only conditionally and blue can not be applied at all.
  • the film is not completely cut and folds during catapulting only, so that no tissue enters the catcher.
  • no automation of the extraction of histological material is possible because it requires constant control whether all catapulting to be catapulted catapults were actually catapulted or have come into the receptacle.
  • the film shows a poor flatness, which is further deteriorated after passing through the histological baths and dyeing solutions, so that in the practical application has to be constantly refocused. This problem is mainly due to the fact that the inner PEN film does not adhere to the slide, so it can not be held in position by the slide, otherwise it would not catapult.
  • the transport system comprises a stable substrate transparent to laser light and a functional layer adhering to this over the entire area (see the unpublished DE 10 2008 026 727 B3).
  • the substrate is preferably a conventional glass slide, and the adhesion between the functional layer and the substrate guarantees a good flatness.
  • the functional layer is between 1 and 10 micrometers thick and is subdivided into at least two sublayers arranged one above the other.
  • a driver layer which has a high absorptivity for the laser light and is capable of locally expanding by laser-induced gas release.
  • a carrier layer is arranged, on which the biological material is applied, whereby the biological material is catapulted together with a carrier layer fragment.
  • US 2007/029 2312 A1 which is to be further specified, also has the task of presenting a sample plate for biological samples, on which the sample material can be examined locally ("addressable analyzes") and, if necessary, isolated at predetermined sampling locations .
  • An addressability of the extraction locations is achieved by pre-installed pallets, which adhere in a defined arrangement on a glass surface. These pallets can be detached, for example, by a laser pulse, which is focused in the interface between glass surface and pallet and forms a plasma there. The pallet is therefore necessarily isolated together with the sample material. The detached pallet is merely transferred to a liquid flow for transport.
  • sample carrier requires some manufacturing effort and yet allows no removal of sample areas that you want to isolate when inspecting a flat sample material (eg histological section) at any point, if no suitably arranged Pallet is present.
  • PEN film technology and functional layer method are suitable for selecting and transporting dissectates that are significantly larger than the laser-irradiated area. In both cases, most of the biological material is unaffected by the laser radiation, so that, above all, the transport of living cells or cell clusters is possible.
  • 6,291,796 B1 which describes a method for cleaning objects by means of pulsed laser radiation (“laser dry cleaning")
  • laser dry cleaning a method for cleaning objects by means of pulsed laser radiation
  • the impurities present on an object surface - which are not seldom organic - are transmitted through a combination of laser ablation, photoinduced decomposition, and laser pulse induced vibration of the object, wherein the object is thermally expanded by linear absorption of the laser pulses.
  • the starting point of the invention is that the transparent substrate itself - commonly a glass slide having two extended flat sides - is to be caused to drop the arranged on a flat side of biological sample material at selected points and to spin into the receptacle.
  • the selection of where to shed this is done by focusing a pulsed laser beam near the sample material.
  • the sample material should be protected from the laser light, so that a radiation from the flat side with the sample is not appropriate. Rather, the laser light must be radiated from the side facing away from the sample material flat side of the substrate and apparently expedient to pass through the substrate largely before it comes to an energy deposition in the vicinity of the sample material. The energy must also be deposited at a minimum distance in front of the sample material in order to avoid the damage caused by heat and radiation.
  • the laser radiation must therefore be focused inside the transparent substrate, so that the energy deposition takes place by non-linear absorption with simultaneous plasma formation.
  • plasma formation irreversibly damages the substrate.
  • the area of damage can be limited to the interior of the substrate (analogous to the laser internal engraving) or continue to the substrate surface with the sample material. It can also lead to fragmentation of substrate fragments.
  • the extent of substrate damage is not critical to the invention.
  • the generation of a plasma in the laser focus under the substrate surface with the sample material entails the emission of a shock wave.
  • the shock wave propagates spherically from the laser focus and, in particular, passes through the predetermined distance between laser focus and substrate surface, whereby the pressure weakens, which is initially very high in the laser focus.
  • the gap not only protects the sample material from the very high plasma temperature, it also allows for "fanning out” of the pressurization of the sample material by the expanding shockwave.
  • a momentum transfer to the sample material occurs. which, given suitable laser parameters, is sufficient for detaching, tearing off and accelerating a piece of the sample material in the direction of the collecting vessel, the sample surface thus transported being much larger than the cross section of the laser focus.
  • the method according to the invention is currently the only method for laser-induced material transport that relies solely on energy deposition by means of non-linear absorption. This has another serious advantage: one is largely free to choose the laser wavelength. In particular, all wavelengths from the UV to the NIR spectral range are suitable for transport. NIR wavelengths are particularly interesting because they are hardly absorbed by biological material. The invention will be explained in more detail with reference to figures. Showing:
  • FIG. 1 shows a schematic cross-sectional view of different phases of the processes in the transparent substrate loaded with sample material when carrying out the method according to the invention
  • Fig. 3 Scanning electron micrographs of the surface of a glass substrate, in which have formed by focusing a laser pulse under the substrate surface different types of damage to the substrate surface.
  • FIG. 4 shows a diagram with experimental measurements of the laser pulse energy required for transporting a specific area of sample material as a function of the distance between laser focus and substrate surface, in which different samples are transported and the possible damage to the substrate surface is examined;
  • Figure 5 is a bar graph of the number of PCR cycles required for a given signal strength for a housekeeping gene comparing two prior art methods (catapulting with PEN foil and catapulting directly from the jar).
  • Fig. 6 is a bar graph analogous to FIG. 5, wherein here for three different
  • the inventive method is compared with the PEN-film technique.
  • the high volumetric energy density within the plasma is associated with the generation of high temperatures of several thousand Kelvin and high pressures in the range of 1 to 10 GPa (Vogel et al., Shock wave emission and cavitation bubble generation by picosecond and nanosecond optical breakdown in water , J. Acoust, Soc., Am., 100: 148-165, 1996.)
  • the maximum pressure amplitude decreases with increasing radius.
  • a shock wave has a particle velocity u p, which is dependent on the pressure jump P at the shock front and the pressure curve within the shock wave, and accordingly also an impulse.
  • the momentum per unit area of a spherical shockwave of radius r is (CoIe, "Underwater Explosions", Princeton University Press, Princeton, New Jersey, 1948, p.112). where t 'corresponds to the shock wave duration.
  • the shock wave is reflected on the free surface of the substrate (or on the sample on it) as a tensile wave with amplitude - P at the shock front.
  • the material surface becomes velocity-dependent within the rise time of the shock front (CoIe, "Underwater Explosions", Princeton University Press, Princeton, New Jersey, 1948, p. 54).
  • u g 2u p cosa accelerated, where a is the angle of incidence of the shock wave (angle between wavefront and substrate surface or between propagation direction and surface normal, a - 0 ° for vertical incidence). This speed is also transferred to the material which is located on the substrate surface.
  • the tensile wave breaks up the substrate material if it exceeds its tensile strength at any location in the material ("spallation"). If the tensile strength is not exceeded, the passage of the shock wave results in reversible transient elastic deformation the substrate surface in shock wave reflection by the elastic restoring forces in the material back.
  • Separation of the sample occurs in the region where the spallation force component directed perpendicular to the surface exceeds both the adhesion to the substrate and the tensile strength to adjacent sample parts.
  • substrate material can be broken out in the area above the plasma, wherein in the area directly above the plasma, the spallation effect is facilitated by the melting of the material.
  • Molten substrate material can pass from the immediate vicinity of the plasma to the surface of the generated crater due to the high pressure in the plasma region or to the substrate surface in the vicinity of the crater. However, since this occurs significantly after detachment of the sample from the substrate surface, the sample material can not be thermally damaged by the melt.
  • the above discussion illustrates the analogy of the inventive method to an underground blast.
  • the laser light is focused inside the transparent substrate at a predetermined distance from the substrate surface ("depth of burial") .
  • depth of burial the biological material on the substrate surface is hardly affected by the laser radiation far outside the laser focus
  • Absorption corresponds to the remote ignition of an explosive device.
  • the substrate material located between the laser focus and the surface acts as a mediator of an impulse transfer driven by the plasma expansion to the biological material arranged on the substrate surface, which is to be sampled.
  • the substrate material also serves as a protective layer against thermal damage to the sample.
  • At least a portion of the biological material is lifted off the substrate and transported to a collection vessel, optionally together with broken fragments of the substrate material. It can create a crater on the substrate surface.
  • the substrate is irreversibly damaged - at least in the interior.
  • the maximum depth of the ejection crater is achieved with brittle materials such as rocks or glass, when spallation and acceleration due to the gas pressure with equal proportions contribute to the formation of craters.
  • the substrate material In the case of ejection of substrate material, this is transported together with the sample into the collecting vessel. If the substrate material is inert to the biochemical processes relevant to genomic or proteomic analysis, the mixing of sample and substrate material will not adversely affect the analysis result. This is the case, for example, with glass and with several polymers (e.g., PEN). For the method according to the invention, it is therefore not essential whether fragments of the substrate are also transported or not.
  • the method has high stability to variations in process dynamics, with the result that it can be used with very different irradiation parameters.
  • the size of the sample area transported corresponds to the area where the momentum transfer to the sample is sufficiently large to detach it from the adjacent sample material, to overcome the adhesion to the substrate surface, and to accelerate it to a startup speed that opposes the distance to the collection vessel Gravitational force and air friction sufficient.
  • a minimum speed w min of the sample can be defined as the transport threshold.
  • the distance of the laser focus to the substrate surface should preferably always be greater than the desired radius of the transported sample area.
  • the minimum distance between laser focus and substrate surface must be at least so large that the sample can not be damaged by heat conduction. This is included
  • Pulse durations in the nanosecond range at a distance of 3 microns certainly the case.
  • the minimum distance must be so large that the beam cross section at the specimen and the specimen surface acted upon by the shock wave are significantly larger than the focal diameter. This condition is fulfilled if the distance is at least equal to the Rayleigh length Z R , within which the beam cross-section doubles. If one looks at the approximation of a Gaussian beam, which is usually permissible for lasers, the Rayleigh length can be expressed as follows
  • CO 0 is the beam radius in focus and ⁇ is the wavelength of the light used.
  • Z R ⁇ 3 microns may be, the minimum distance for the transport according to the invention nevertheless - as explained above - be 3 microns.
  • the laser wavelength must fall within the transmission range of the substrate material, which ranges, for example, in glass from the UV-A region to the near infrared region of the optical spectrum. Since the energy deposition takes place by non-linear absorption and plasma formation, which has only a low wavelength dependency, in principle all laser wavelengths in the transmission range of the substrate material are suitable. This is an advantage over the prior art methods in which the linear absorption properties of the sample material and the particular layer system used must be taken into account.
  • the ability to use IR wavelengths, such as the fundamental wavelength of the Nd: YAG laser (1064 nm) may be additional Protecting the transported biological samples, the linear absorption is usually much lower in the near IR than at UV wavelengths.
  • Particularly advantageous and inexpensive is the use of pulses of microchip lasers having a duration between about 300 ps and 3 ns.
  • the laser beam quality must be as good as possible in order to minimize the energy threshold for plasma formation in the laser focus.
  • K - MM 2 and diffraction factor have the value 1, for real laser beams M 2 > 1 and K ⁇ 1.
  • Microchip lasers usually emit radiation with a diffraction factor M 2 ⁇ 1.3, and are thus excellently suited for the task ,
  • nitrogen lasers are commonly used for dissection and catapulting.
  • the jet properties of nitrogen lasers are so poor that in the substrate material at the moderate numerical apertures commonly used for catapulting (10x and 2Ox lenses with - -
  • NA ⁇ 0.5 can not realize an optical breakthrough. Due to the absence of an optical breakdown in the substrate, the laser energy is deposited directly in the sample and the transport takes place, as already described in the prior art, by evaporation of a portion of the sample. This is accompanied by damage to the sample. Nitrogen lasers or other laser types with poor jet properties are therefore not suitable for the process according to the invention.
  • the numerical aperture (NA) of the objective used to use the laser radiation may be in the range 0.2 ⁇ NA ⁇ 1.3.
  • Numerical apertures up to 0.75 can be realized according to the prior art by means of long-distance lenses with correction compensation in conjunction with 1 mm thick glass substrates.
  • To realize larger apertures up to NA 1.4, oil immersion objectives in combination with a cover glass of about 0.1-0.2 mm thickness must be used as the substrate.
  • the minimum laser pulse energy for the laser-induced transport is given by the at least required energy threshold E ⁇ n for the plasma formation within the substrate material. This is defined by creating a permanent damage / defect in the substrate material.
  • the defect produced by plasma formation always reaches all the way up to the substrate surface.
  • the minimum energy can be easily determined experimentally by subsequent surface inspection.
  • the pulse energy is to be increased if the laser focus is selected further away from the substrate surface.
  • preliminary experiments should be performed for each particular choice of substrate and sample material, in particular varying pulse energies and depth of focus, to seek out the area of optimal sample transport.
  • the minimum lateral distances between the individual laser exposures in the scanning of the biological sample arise from the region of the sample, where the momentum transfer by shock wave and / or broken substrate material is sufficiently large to reproducibly detach the sample from the surrounding sample material and towards the collecting vessel accelerate.
  • Large distances between the halftone dots in conjunction with a high repetition rate of the laser pulses accelerate the speed of sample collection.
  • the spacings of the halftone dots should preferably not be greater than twice the distance of the laser focus from the substrate surface with the sample material.
  • FIG. 1 shows schematic cross sections through a transparent substrate (dotted) with a film of sample material arranged thereon (interrupted hatching), which adheres only relatively weakly to the substrate.
  • the arrow indicates the direction of irradiation of the laser light
  • the cone lines indicate the beam cross-section, which is minimal in the laser focus (dark oval).
  • the laser focus has a clear distance from the substrate surface.
  • Fig. 1 b is a snapshot of the propagation of a shock wave to see, which was previously triggered by plasma formation in the laser focus.
  • the shock wave initially spreads out spherically. If it reaches a free surface, it is reflected there and converted into a tensile wave, whereby a pulse is transmitted. Acceleration of the sample material occurs where the momentum transferred to the sample film by the impact is sufficient to overcome the adhesion of the sample to the substrate.
  • FIG. 1 b it is assumed in a simplified manner that the acoustic impedance in substrate and sample material are identical. This is usually not the case, so that the shock wave is largely reflected already at the substrate / sample interface. Nevertheless, a transfer of the momentum to the sample is also possible by the movement of the substrate surface.
  • the detachment and lifting of a portion of the sample film as shown in Fig. 1 c is carried out when the momentum transfer region is large enough, in addition to the adhesion to overcome the cohesion of the sample film. This is indicated by jagged crack edges in the sample film.
  • the lifted sample piece is accelerated in the direction of the collecting vessel.
  • maximum sample radius b max can be lifted perpendicular to the substrate surface at a predetermined focus depth d in the forward direction.
  • the substrate S is indicated solely by its surface (thick line), and the laser focus P is located at a distance d to this surface in the interior of the substrate.
  • the edge region of the sample is precisely characterized by the condition that the shockwave front there encloses the angle a R with the substrate surface. As already mentioned, it is just there (see equation (4))
  • the depth of the laser focus in the substrate should therefore be selected to be greater than the desired radius of the transported sample area. It should be noted, of course, that one has to tune the focus depth and the laser pulse energy suitable for each other when specifying the sample radius, so that a sufficient momentum transfer to the sample is possible.
  • FIG. 3a shows a scanning electron micrograph of a crater in the substrate after such a fragment has been removed.
  • the morphology of the visible damage can vary considerably with the depth of focus, as shown in FIGS. 3b and c by way of example.
  • FIG. 4 illustrates the distance of the laser focus to the substrate surface.
  • the laser pulse energy required for successful transport is plotted on the ordinate.
  • a transport experiment is defined as successful if, during the lateral displacement of the laser focus, a strip of the sample material with a defined width can be completely transported. This implies both the repeatability of single-pulse transport and at the same time examines the convenience of the procedure for scanning sample areas, which is believed to be the main application of the invention.
  • biological samples are pulsed with pulsed laser light scanned by a carrier, and the collected samples are RNA extraction and analysis by quantitative "reverse transcription polymerase chain reaction” (qRT-PCR) supplied.
  • qRT-PCR quantitative reverse transcription polymerase chain reaction
  • Fig. 5 shows first exclusively the prior art. In this experiment, pulses of a nitrogen laser with about 10 ⁇ J of energy are applied through a 10x objective lens on the
  • FIG. 6 shows the comparison of the method according to the invention (dark hatched) with the PEN-film technique (bright hatching) for three different housekeeping genes, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-phosphoribosy 1-transferase ( HPRT), and Metastatic Lymph Node 51 (MLN51).
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • HPRT hypoxanthine-phosphoribosy 1-transferase
  • MN51 Metastatic Lymph Node 51

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

La présente invention concerne un procédé de transport induit par laser d'une matière dans un récipient collecteur au moyen d'un rayonnement laser pulsé, la matière étant disposée sur un côté d'un substrat transparent au rayonnement laser, et le rayonnement laser étant introduit dans le substrat depuis un côté de celui-ci opposé à la matière. Le procédé comprend les opérations suivantes : focalisation du rayonnement laser pulsé à l'intérieur du substrat de sorte que le foyer laser présente une distance prédéterminée par rapport au côté du substrat chargé de matière; production d'un plasma dans le foyer laser par absorption non linéaire du rayonnement laser; et arrachement et accélération de la partie de matière la plus proche du foyer laser en direction du récipient collecteur, par transmission des impulsions d'une onde de choc qui se propage depuis le foyer laser et parcourt la distance prédéterminée jusqu'au côté du substrat chargé de matière.
PCT/DE2009/001297 2008-09-23 2009-09-14 Procédé de transport induit par laser de matières depuis un substrat de support transparent Ceased WO2010034289A1 (fr)

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DE102008048618.3 2008-09-23
DE102008048618A DE102008048618B3 (de) 2008-09-23 2008-09-23 Verfahren zum laser-induzierten Transport von Materialien von einem transparenten Trägersubstrat

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US20220276136A1 (en) * 2021-03-01 2022-09-01 Leica Microsystems Cms Gmbh Method for obtaining dissectates from a microscopic sample, laser microdissection system and computer program
US12038357B2 (en) * 2021-03-01 2024-07-16 Leica Microsystems Cms Gmbh Method for obtaining dissectates microscopic sample, laser microdissection system and computer program

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