WO2008000389A1 - Procédé et dispositif pour traiter des objets biologiques à l'aide d'un rayonnement laser - Google Patents

Procédé et dispositif pour traiter des objets biologiques à l'aide d'un rayonnement laser Download PDF

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Publication number
WO2008000389A1
WO2008000389A1 PCT/EP2007/005485 EP2007005485W WO2008000389A1 WO 2008000389 A1 WO2008000389 A1 WO 2008000389A1 EP 2007005485 W EP2007005485 W EP 2007005485W WO 2008000389 A1 WO2008000389 A1 WO 2008000389A1
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WO
WIPO (PCT)
Prior art keywords
treatment
shaping element
laser
type
profile
Prior art date
Application number
PCT/EP2007/005485
Other languages
German (de)
English (en)
Inventor
Yilmaz Niyaz
Karin SCHÜTZE
Carsten LÜTHY
Original Assignee
P.A.L.M. Microlaser Technologies Gmbh
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by P.A.L.M. Microlaser Technologies Gmbh filed Critical P.A.L.M. Microlaser Technologies Gmbh
Publication of WO2008000389A1 publication Critical patent/WO2008000389A1/fr

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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
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G01N2001/045—Laser ablation; Microwave vaporisation
    • 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 present invention relates to a method and a device for the treatment of biological objects by means of laser radiation, for example for laser microdissection, for laser pressure catapulting (LMPC) and / or for laser-assisted micromanipulation.
  • laser pressure catapulting also referred to as catapulting for short in the following
  • biological objects can be at least largely cut out of a biological mass and then catapulted with a targeted laser pulse into a collecting container, which presumably depends on the ignition of a microdissection. plasmas is due.
  • Direct catapulting of regions of interest of the biological mass without prior excision is also possible, in which case the catapulted region is critically dependent on the beam intensity and the beam profile of the laser beam used.
  • biological objects e.g. Objects in a liquid
  • optical tweezers biological objects are fused together, and / or manipulation or cutting processes are performed within cells.
  • a device or such a method is known, for example, from DE 100 15 157.4 of the Applicant.
  • Such systems conventionally use the same laser for the various types of biological object treatment, for example both cutting and catapulting.
  • the beam profile generated by the laser is used essentially unchanged, both for the microdissection process and for the catapult process.
  • the laser parameters are usually set for the respective procedure and for the particular preparation: For example, during the microdissection process, the focal point of the laser is at the object plane, while the laser beam is used defocused for the catapulting process.
  • the laser energy between the cutting edges changed in the microdissection and catapulting, for example, increased. This results in a strong dependency of the catapulting on the quality and geometry of the output beam: For the cutting in the microdissection, beam profiles with a narrow Gaussian distribution are suitable, which however are not optimal for catapulting.
  • the above-mentioned defocusing which can be done for example via a beame-pander and gray-scale attenuator, produces a compromise between the opposing requirements of cutting and catapulting.
  • the "transfer pulse” is set via defocusing of the laser beam, with the result that a large part of the available energy does not benefit the transfer, ie the catapulting process, but, for example, only the basis of the gaussian process.
  • the residual energy is either lost underneath the object plane, or transferred to the object plane, resulting in unnecessary load on the sample (penetration of the sample), resulting in high-numerical objectives for difficult-to-process samples for the microdissection process
  • some types of lasers such as femtosecond lasers
  • catapulting is difficult and can only be carried out to a limited extent since the energy transferred only has a very small surface area ve can be given.
  • a method for the treatment of biological objects according to at least a first type of treatment and a second type of treatment different from the first type of treatment wherein the biological object is irradiated with a laser beam, and wherein at least for treatment according to the first treatment or treatment according to the second treatment Beam shaping element is moved into the beam path of the laser beam.
  • a beam-shaping element generally designates an optical element with which the profile, i. the intensity distribution of the laser beam can be modified.
  • the profile of the laser beam can be of the appropriate type of treatment, e.g. cutting in laser microdissection, catapulting or laser assisted micromanipulation.
  • no beam-shaping element can be moved into the beam path for the first type of treatment, and a beam-shaping element can be moved into the beam path for the second type of treatment.
  • a first beam-shaping element is moved into the beam path for the first type of treatment, while a second element is moved into the beam path for the second type of treatment. It is also possible to provide other types of treatment, if necessary in conjunction with further beam-shaping elements.
  • the beam-shaping element is configured to form a beam having a Gaussian profile into a beam having a non-Gaussian profile, for example a beam having a plateau-shaped profile
  • Profile reshaped. With gaussförmigem profile, which in one embodiment Then, for example, a cutting process may be performed while using a plateau-shaped profile for a catapulting process.
  • the beam-shaping element is configured to divide a single laser beam (primary beam) into a plurality of laser beams (secondary beams), which are then simultaneously used to treat the biological object, e.g. for catapulting, can be used.
  • the laser beam used can be adapted very quickly to the respective type of treatment. Depending on the application, this may be due to changes in focusing or beam energy between types of treatment, e.g. between dissection and catapulting, and / or a change of objective.
  • the energy input into the sample can be minimized, which can reduce the damage to the sample, for example in living cells. It is also possible in this way to prevent the penetration of a membrane used which carries the samples.
  • a predetermined area can be achieved without cluttering. Overlap areas are irradiated, which is helpful for the above-mentioned direct catapulting without prior dissection.
  • FIG. 1A shows a beam path of a laser in an embodiment of a device according to the invention
  • FIG. 4 shows intensity distributions of exemplary embodiments of beam shaping elements according to the invention
  • 5A and 5B show a phase structure of a further embodiment of a beam shaping element according to the invention
  • FIGS. 8 and 9A and 9B are diagrams for illustrating the production of beam shaping elements according to the invention.
  • FIG. 10 shows an embodiment of a system with a beam-shaping element according to the invention.
  • FIGS. 1A and 1B show an exemplary embodiment of a device according to the invention, wherein FIG. 1A shows the beam path and FIG. 1B shows a complete microscope system.
  • the microscope system of Figure 1 B is based on a conventional system for the treatment of biological objects, in particular for laser microdissection and laser pressure catapulting (LMPC), as described for example in DE 103 58 565 of the applicant.
  • LMPC laser microdissection and laser pressure catapulting
  • the system shown in FIGS. 1A and 1B comprises a laser device 17 in which a laser 120, for example an ultraviolet laser, is accommodated for generating a laser beam.
  • a laser 120 for example an ultraviolet laser
  • an optical system 16 is accommodated in the laser device 17, by means of which the laser beam can be coupled into a microscope 13 and the laser focus in the object plane can be matched to the optical focus of the microscope 13.
  • a control panel may be provided with the aid of which the laser energy and / or the laser focus can be set to desired values.
  • the laser focus can also be set independently of the microscope focus by means of the optics 16, ie the focal point of the laser can be displaced in the z-direction relative to the object plane of the microscope 13.
  • the laser beam For coupling the laser beam into the microscope mirror or beam splitter 15 are further provided by which the laser beam is deflected to a lens 12 out.
  • the laser beam emitted via the objective 12 finally impinges on a motorized and computer-controlled microscope or support stage 14, for example, on which a specimen slide with a biological mass to be processed can be arranged.
  • a motorized and computer-controlled microscope or support stage 14 O- above the support table 14 is also a motorized and preferably computer-controlled collecting device 19, which one or more Receiving or collecting elements or collecting vessel 1 has.
  • the components 14 and 19 enable exact object positioning as well as precise collection of biological objects which are cut out of the mass located on the support table 14 by laser irradiation and / or are catapulted outwards.
  • the microscope 13 may be an arbitrarily designed microscope. In particular, the use of both an inverted microscope as shown in Fig. 3 and an upright microscope or a laser microscope is conceivable in principle.
  • the microscope 13 is configured in one embodiment with a video camera, which receives the area of the slide or support table 14 above the lens 12.
  • the video signal of this video camera is supplied to a commercially available computer 18 and there subjected to such image processing that the corresponding video image can be displayed in real time on the screen or monitor 8 of the computer 18.
  • Various functions are implemented in the computer 18 or the software running on it, which enable both a computer-aided, ie automatic, control of the laser device 17 and the microscope 13 or the support table 14 and the catching device 19, so that, for example, the laser is automatically activated and the collecting device 19 and the support table 14 automatically moved and can be adjusted.
  • conventional input means such as a keyboard 9, a computer mouse 10 or the like are provided.
  • the laser device 17 is assigned a foot switch 11, by the actuation of which the laser can be activated manually.
  • further optical elements 20 (not shown in FIG.
  • the laser beam may be directed via an optical system designated by reference numeral 21 in FIG. 1A which also serves to illuminate a sample located on the support table 14.
  • the UV laser 120 may emit a gaussian laser beam of 355 nm wavelength.
  • the waist diameter of the laser beam may be on the order of 250 ⁇ m.
  • a beam-shaping element 100 which can be driven by a stepping motor as indicated by an arrow 110 into the beam path or can be driven out of it serves.
  • a stepping motor instead of a stepping motor, other means of movement are also conceivable for moving the beam-shaping element 100 into the beam path or out of the beam path.
  • the stepping motor preferably has a high positioning accuracy in order to be able to precisely set the beam profile or to be able to precisely position the beam-shaping element 100.
  • One possible positioning accuracy is, for example, 0.1 mm.
  • the controller can be made for example by the computer 8.
  • the position of the beam-shaping element 100 shown in FIGS. 1A and 1B serves merely as an example, and in other exemplary embodiments such a beam-shaping element can also be positioned at a different location in the beam path of the laser.
  • a plurality of beam-shaping elements can be provided, which can be moved into the beam path individually or in groups depending on a particular application or type of treatment.
  • two different beam-shaping elements 100 are also conceivable, one being moved into the beam path for cutting and one for a catapulting operation in order to generate an optimum beam profile in each case.
  • the beam-shaping element 100 may in particular be or contain a diffraction element (diffractive element). Exemplary embodiments of beam-shaping elements were explained below.
  • the diffraction element can be, for example, a so-called asphere.
  • an aspheric is understood to mean an aspherical diffraction element with a diffraction grating or another diffraction profile, which can be produced, for example, by lithography as shown in FIGS. 9A and 9B.
  • desired beam profiles can be generated.
  • the desired beam profile is specified and the necessary diffraction profile is determined according to the known laws of wave optics.
  • Such diffraction elements are also known, for example, from DE 102 45 558 A1.
  • aspherical diffraction elements can also consist, for example, of a combination of a spherical or aspherical lens with a diffraction grating applied to the lens, so that the beam shaping element in this case forms the beam profile both by diffraction (diffraction) and refraction (refraction).
  • a substrate 30, for example a quartz substrate is provided with a lacquer layer 31 and illuminated.
  • a paint profile is a blazed surface profile for high spatial sequences. Due to the limited profile depth in the photoresist, an additional ion etch process can be used to set the profile depth.
  • the stepped lacquer profile results in points of minimal intensity in transmission, which results in a beam profile when the diffraction element radiates, which is dependent on the lacquer profile of the development 33.
  • the paint profile thus forms a diffraction grating.
  • the skid tion of passing through the resist structure beams by diffraction and / or refraction, whereby a desired beam profile can be generated.
  • such an asphere is configured to act as a converging lens in a central area such as a diverging lens and in an edge area surrounding the central area.
  • FIG. 2A shows a representation of the real part of the phase structure of an embodiment of such an asphere
  • FIG. 2B shows a section along an arrow 40 of FIG. 2A.
  • Figs. 3A-3H show intensity distributions of an output-side beam profile, i. the intensity distribution perpendicular to the beam direction of such an asphere when illuminated with a laser beam with gaussförmigem beam profile.
  • 3A, 3C, 3E and 3G show three-dimensional intensity distributions, the intensity, i. the square of the amplitude expressed by the gray scale.
  • FIGS. 3B, 3D, 3F and 3H show sections along arrows 41, 42, 43 and 44 of FIGS. 3A, 3C, 3E and 3G, respectively.
  • the figures show the intensity distribution, i. the beam profile in an object plane of a corresponding microscope setup with different focussing.
  • the focus is 80 ⁇ m below the object plane
  • Fig. 3C and 3D is focused in the object plane
  • Figs. 3E and 3F the focus is 80 ⁇ m above the object plane.
  • an approximately plateau-shaped beam profile can be generated by a beam-shaping element.
  • approximately plateau-shaped means that the beam intensity varies by less than 30% over a plateau width b1, while at the edge of the plateau within a waste area b2, which is, for example, ⁇ 60% of the plateau width b1, drops to 10% of the maximum intensity.
  • the absolute plateau width depends on the focusing, for example.
  • FIGS. 3G and 3H show the intensity distribution of the beam profile, in the case of a decentered asphere.
  • the asphere is decentered by 0.1 mm with respect to the beam axis of the incident laser beam.
  • the plateau also becomes symmetrical.
  • Figure 4 shows the influence of a tread depth of the asphere, e.g. a depth of the profile shown in Fig. 9B, on the intensity distribution.
  • a curve 50 shows the intensity profile of a gaussian laser beam which strikes the asphere.
  • Curve 51 shows the output intensity profile at a reference profile depth
  • curve 52 shows the output profile at a profile depth increased by the factor 1, 2 with respect to the reference depth
  • curve 53 shows the intensity profile at a profile depth increased by a factor of 1.4.
  • an asphere is used as the beam-shaping element 100.
  • an array is used as the beam shaping element, for example a lens array or a holographic array.
  • Such holographic arrays are, for example, so-called computer-generated hologram arrays (CGH arrays). Arrays can be used in particular with low or no coherence lasers.
  • FIG. 10 An arrangement with a lens array 61 having a plurality of lenses 62 is shown schematically in FIG.
  • the lens array 61 is illuminated with a collimated laser beam 55 via a first stage mirror 57 and a second stage mirror 59. While in front of the step levels, the plane of equal emission time as indicated by a rectangle 56 If a single plane perpendicular to the beam is interpreted as being a single plane perpendicular to the beam, this changes to a plurality of sub-levels 58 after the first level mirror 57 and to a plurality of sub-levels 60 after the second level mirror 59, since different transit times are achieved by reflection at different stages.
  • such a lens array comprises a plurality of nominally identical lenses, e.g. Rectangular lenses.
  • a laser beam primary beam
  • secondary beams each passing through a lens.
  • FIGS. 5A and 5B show a detail of the structure of a CGH array according to an embodiment of the invention, FIG. 5A showing a plan view and FIG. 5B a three-dimensional view. In both Figures 5A and 5B, the profile height of the array is indicated by gray levels.
  • the height scaling of such a profile may be, for example, ⁇ 40 nm.
  • FIGS. 6A and 6B intensity distributions of a laser beam modified by such an array are shown, similar to Figs. 3A, 3C, 3E and 3G.
  • 6A shows the intensity distribution for focusing in the object plane
  • FIG. 6B shows the intensity distribution for focusing 80 ⁇ m above the object plane.
  • the resulting beam profile comprises a plurality of maxima corresponding to a plurality of secondary beams whose width and shape depends on the focusing.
  • the laser energy is distributed over a wider area.
  • the envelope of the maxima yields approximately a plateau, for example a plateau in the sense already described for the asphere.
  • each secondary beam then generates a microplasma, which contributes to the catapult effect.
  • the regular arrangement of the lenses or holographic elements can lead to interference phenomena.
  • a so-called statistical array can also be provided as the beam-shaping element 100, which enables a field distribution substantially without interferences, since in such a statistical array there is no fixed phase relationship between a plurality of beams necessary for an interference.
  • Such statistical arrays are described, for example, in L. Erdmann et al., "MOEMS-based Lithography for the Fabrication of Mirco-Optical Components", Journal of Microlithography, Microfabrication and Microsystems, Vol. 4, Issue 4, 2005.
  • An embodiment of such Statistical arrays include a plurality of lenses, for example rectangular lenses, or holographic elements of different sizes and / or with different optical properties, which are arranged statistically distributed.
  • Figures 7A-7R show far-field intensity distributions for different focuses of such a statistical array.
  • 7A-7C show the intensity distribution for a z-factor of 0.8, FIGS. 7D-7F for a z-factor of 0.9, FIGS. 7G-7I for a z-factor of 1, 0, FIG 7J-7L for a z-factor of 1, 1, Fig. 7M-7O for a z-factor of 1, 2 and Fig. 7P-7R for a z-factor of 1, 3.
  • a z-factor of 1, 0 corresponds to a focus in the object plane.
  • the two-dimensional beam profile is shown as a gray-scale representation, the intensity being represented by the color, and two mutually perpendicular sections (denoted Y-view and X-view).
  • the area considered is 6-6 mm 2 , with the scale given in mrad, that is, as the viewing angle.
  • the intensity distributions are each normalized such that the maximum intensity is 1.
  • wide beam profiles can be produced, which have several maxima.
  • the formation of an approximately plateau-shaped beam profile is therefore also possible with statistical arrays.
  • DMD elements can be used, which are known from projection technology. This involves a large number of micromirrors, which may be arranged in a matrix, and which can be controlled individually.
  • FIGS. 8A-8C show a wafer 70, for example a 4-inch silicon wafer (corresponding to 100 mm diameter), on which a multiplicity of DMD fields 71 are arranged.
  • the individual DMD fields can be, for example, 8.3-8.3 mm 2 in size, the gaps being between 2 and 3 mm.
  • FIGS. 8B and 8C show various ways of dividing the wafer 71 into individual optical elements each having a plurality of DMD fields by sawing along saw lines 72.
  • the beam-shaping element 100 may also consist of a plurality of optical elements, for example a combination of diffractive elements and lenses. Also, combinations of the beam shaping elements 100 discussed above, i. Combinations of aspheres and / or arrays, optionally with other optical elements, are possible.
  • beamforming elements designed to produce a beam profile that is designed for other types of biological object treatment than laser microdissection and catapulting, for example for laser-assisted micromanipulation, may also be used.
  • a beam shaping element can be used which laterally confines a laser beam, i. produces a very narrow beam profile.

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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)
  • Laser Surgery Devices (AREA)
  • Laser Beam Processing (AREA)

Abstract

L'invention concerne un système laser pour traiter des objets biologiques avec un élément de formation de rayons (100).
PCT/EP2007/005485 2006-06-30 2007-06-21 Procédé et dispositif pour traiter des objets biologiques à l'aide d'un rayonnement laser WO2008000389A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200610030195 DE102006030195A1 (de) 2006-06-30 2006-06-30 Verfahren und Vorrichtung zur Laser-Mikrodissektion und zum Lasercatapulting
DE102006030195.1 2006-06-30

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Publication number Priority date Publication date Assignee Title
DE102007030320B4 (de) * 2007-06-29 2015-04-02 Carl Zeiss Microscopy Gmbh Laser-Mikrodissektionsverfahren und Laser-Mikrodissektionssystem

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US20020185592A1 (en) * 2001-06-06 2002-12-12 Grier David G. Optical peristaltic pumping with optical traps
DE10245558A1 (de) * 2002-09-30 2004-04-08 Carl Zeiss Jena Gmbh Abbildungsoptik
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DE10018255C2 (de) * 2000-04-13 2003-08-28 Leica Microsystems Laserschneid-Verfahren und Laserschneid-Vorrichtung zum Laserschneiden mit mikroskopischer Proben
DE10043504C2 (de) * 2000-09-01 2002-07-04 Leica Microsystems Verfahren zur Laser-Mikrodissektion und Verwendung einer Vorrichtung zur Laser-Mikrodissektion
DE10043506C1 (de) * 2000-09-01 2001-12-06 Leica Microsystems Verfahren und Vorrichtung zur Laser-Mikrodissektion
US6813008B2 (en) * 2002-06-10 2004-11-02 Palantyr Research, Llc Microdissection optical system
JP2004170930A (ja) * 2002-10-31 2004-06-17 Olympus Corp マイクロダイセクション装置および方法
DE102004023262B8 (de) * 2004-05-11 2013-01-17 Carl Zeiss Microimaging Gmbh Verfahren zur Bearbeitung einer Masse mittels Laserbestrahlung und Steuersystem

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Publication number Priority date Publication date Assignee Title
DE10015157A1 (de) * 2000-03-27 2001-10-18 P A L M Gmbh Verfahren zur Bearbeitung einer biologischen Masse und Steuersystem für eine Vorrichtung zur Bearbeitung einer biologischen Masse
US20020185592A1 (en) * 2001-06-06 2002-12-12 Grier David G. Optical peristaltic pumping with optical traps
DE10245558A1 (de) * 2002-09-30 2004-04-08 Carl Zeiss Jena Gmbh Abbildungsoptik
WO2005033669A1 (fr) * 2003-10-02 2005-04-14 Leica Microsystems Cms Gmbh Procede de microdissection au laser

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