EP2181316A1 - Procédé et dispositif de traitement d'un objet biologique avec un rayon laser - Google Patents

Procédé et dispositif de traitement d'un objet biologique avec un rayon laser

Info

Publication number
EP2181316A1
EP2181316A1 EP08774003A EP08774003A EP2181316A1 EP 2181316 A1 EP2181316 A1 EP 2181316A1 EP 08774003 A EP08774003 A EP 08774003A EP 08774003 A EP08774003 A EP 08774003A EP 2181316 A1 EP2181316 A1 EP 2181316A1
Authority
EP
European Patent Office
Prior art keywords
biological object
laser beam
focal planes
biological
different focal
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
Application number
EP08774003A
Other languages
German (de)
English (en)
Inventor
Bernd SÄGMÜLLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Carl Zeiss Microscopy GmbH
Original Assignee
Carl Zeiss MicroImaging 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 Carl Zeiss MicroImaging GmbH filed Critical Carl Zeiss MicroImaging GmbH
Publication of EP2181316A1 publication Critical patent/EP2181316A1/fr
Ceased legal-status Critical Current

Links

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
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
    • 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 present invention relates to a method and an apparatus for processing a biological object by means of laser radiation.
  • a generic device is also known for example from EP 1 269 142 A of the applicant.
  • selected regions of biological objects can be cut out by computer-controlled means of a pulsed laser according to the principle of so-called laser microdissection.
  • a user can specify a cutting line by means of a computer, whereupon a computer-controlled cutting out of the desired region takes place from the biological object or the biological mass.
  • the areas thus cut out can then be catapulted by means of a laser pulse into a collecting container.
  • the device described in EP 1 269 142 A is particularly suitable for processing biological objects which lie planar on a slide and / or are not too thick, so that the laser beam can be focused in a simple manner on the plane of the object and it is sufficient to cut completely through the biological object, with the laser energy selected such that the cut-out area remains usable for subsequent examinations.
  • the device described in EP 1 269 142 A is also suitable for other types of processing of biological masses or objects.
  • fluorescent substances in a biological mass can be excited to fluorescence by means of a suitably focused laser beam.
  • the device is particularly suitable for the treatment of planar located on a slide biological objects of limited thickness.
  • a method for processing a biological object comprises irradiating the biological object with a laser beam and automatically focusing the laser beam for irradiation on at least two different focal planes, wherein the focus of the laser is at least approximately within of the object, eg maximum 20 ⁇ m outside the object.
  • the at least two different levels may include a plurality of different levels at predetermined intervals.
  • a given level grid can be used.
  • the laser focus is at least approximately within the object when it is maximally on the plane of the plurality of planes immediately above or immediately below the object.
  • the method according to the invention is suitable for different types of biological objects, e.g. Tissue sections, cell cultures and the like.
  • the irradiation of the object to cut out a region of interest from the object In one exemplary embodiment, a closed cutting line with the laser is traversed on each at least two different planes, so that a stepwise cutting out of the region of interest can take place from a thicker object.
  • non-closed cutting lines are used on the at least two different planes.
  • information from an imaging process such as the so-called z-stacking can be used.
  • z-stacking an image of the object is taken, for example, by means of a microscope setup in different focal planes of a lens, a resulting image being composed of those parts of the individual images which are in focus.
  • the planes for recording the individual images can comprise the at least two levels in an exemplary embodiment.
  • the laser focus when irradiating the object can be set in each case to that level in which the best focus is present at the corresponding location in the imaging process.
  • the method of the invention can be used not only to excise regions of interest but, for example, to ablate (leach) a portion of the thickness of the object, to excite fluorescence in the biological object, and / or to bleach bleachable absorbers in the biological object. Other applications are possible.
  • a corresponding apparatus for processing an object comprises a laser, an optics for focusing the laser on an object located on the support and a focusing control, which is designed such that it moves the laser beam on at least two different planes parallel to the plane defined by the support focused during the irradiation of the object.
  • FIG. 1 shows an embodiment of a device according to the invention
  • FIG. 2 shows a side view of a biological object for illustrating an embodiment of a method according to the invention
  • FIG. 3 is a plan view of the arrangement of Fig. 2,
  • FIG. 4 shows a further side view of a biological object to illustrate a further embodiment of a method according to the invention
  • 5 is a plan view of the arrangement of Fig. 4th
  • FIGS. 1-5 shows an exemplary embodiment of a device according to the invention for processing biological objects, in the form of a microscope system which is used, for example, for laser microdissection, i. can be used for cutting biological objects, for catapulting by means of laser radiation as well as generally for the manipulation of biological objects by means of laser radiation.
  • the system shown in FIG. 1 comprises a laser device 11 with a laser 18.
  • the laser 18 may be, for example, a frequency tripled neodymium YAG laser. However, other types of lasers such as solid state lasers, gas lasers such as nitrogen lasers or dye lasers are possible.
  • a laser beam emitted by the laser 18 is coupled through an optical system 15, 16 and mirror 17 into a microscope 13 and directed to a microscope objective 12 of the microscope 13.
  • the optics 15, 16 comprises in the illustrated embodiment, a neutral density filter 15 for adjusting the intensity of the laser beam and lenses 16 for focusing the laser beam.
  • this arrangement makes it possible to adjust the focusing of the laser beam both through the microscope objective 12 and through the lenses 16, so that the laser beam can be focused both together with the microscope 12 and independently thereof.
  • the mirrors 17 can each be configured as conventional correspondingly coated mirrors or as beam splitters, for example in the form of prisms.
  • the microscope 13 shown in FIG. 1 is a so-called inverted microscope, in which the microscope 12 is arranged below a support table 14. On the support table 14 is then - for example, on an additional slide - an object to be processed, in particular a biological object or a biological mass arranged.
  • An image recording device 31 is provided in the microscope 13, for example a CCD camera with which an image of an object located on the support table 14 can be recorded via the microscope objective 12.
  • the exemplary embodiment illustrated in FIG. 1 has a collecting device 10 in which, for example, regions cut out of a biological object by means of the catapulting method mentioned in the introduction or in another way can be transported.
  • the collecting device 10 can be omitted.
  • an inverted microscope as shown, an upright microscope can also be used in which the viewing of an object located on the support table and / or irradiation of the object with the laser beam occurs from above the support table.
  • the apparatus shown in Fig. 1 is controlled by a computer 21 having, in the illustrated embodiment, a data output screen 22 and a keyboard 19 and a mouse 20 for inputting data. It can also be provided more peripherals.
  • the computer 21 controls in the embodiment in particular the laser device 11 with the lenses 16, the support table 14, which may be configured as a motorized xy-table, the neutral density filter 15 and optionally the catching device 10.
  • the computer 21 controls the microscope objective 12, which can be moved in the illustrated embodiment for focusing in the z-direction.
  • the plane of the support table 14 is assumed to be the xy plane, while the direction perpendicular to it is the z direction.
  • these names are only for indication of direction and can be chosen differently.
  • the laser beam before it hits an object lying on the support table 14, thus runs in the z-direction.
  • the device illustrated in FIG. 1 can be cut out, for example, from a biological object located on the support table 14 by means of the laser beam emitted by the laser 18, an area of interest.
  • a cutting line are set, which is then traversed automatically.
  • FIG. 2 shows a side view of a biological object 30 located on a slide 23.
  • FIG. 3 shows a corresponding plan view.
  • slide 23 may be a laser beam 31 emitted by lasers 18, a transparent glass slide, or a membrane slide placed on support table 14 of FIG.
  • a biological object directly on the support table or, for example, in a container such as a petri dish, on the support table 14.
  • the biological object 30 has a thickness that is so large that it can not readily be cut in one pass with the laser beam 31.
  • the laser power is limited by the fact that although a cutting process to be performed, but the cut-out area and / or the non-cut portion of the biological
  • Object should be changed as little as possible. This results, for example, in a limitation of the section thickness of the order of magnitude of 20 .mu.m, which value may vary depending on the object to be processed and the laser used.
  • an area is to be cut out, which is outlined by a section line 27, 28, 29 in Fig. 3.
  • a cutting line can be entered by means of the computer 21 by a user.
  • the cutting is carried out successively in different focal planes 24, 25, 26, which each have a distance ⁇ z.
  • the illustrated three levels 24, 25, 26 are to be understood merely as an example.
  • more or less than three focal planes may be used.
  • ⁇ z can be 5 ⁇ m, so that 20 focal planes are used.
  • each focal plane of the laser beam is in the embodiment of FIGS. 2 and 3 along a closed section line as shown in Fig. 3 moves.
  • a cut line 27 is drawn in the focal plane 24
  • a cut line 28 is drawn in the focal plane 25
  • a cut line 29 is drawn in the focal plane 26.
  • the biological object 30 is cut through step by step.
  • the microscope objective 12 is moved in the z-direction in one exemplary embodiment.
  • the object carrier 23 is moved in the z-direction, for example by movement of the carrier table 14 from FIG. 1, in order to change the focal plane.
  • lenses within or outside the microscope objective may be moved to adjust the focal plane.
  • the object 30 For tracing the cutting line in the xy plane, ie in the plane of the slide 23 and the planes 24, 25, 26, the object 30 is then moved relative to the laser beam 31, for example by an xy movement of the support table 14 of FIG. 1.
  • the distances between the levels are constant.
  • the distance may have a predetermined value, which is used independently of the object 30.
  • the distance .DELTA.z can also be determined depending on the object used.
  • the distances between the focal planes may also vary.
  • FIGS. 4 and 5 show a biological object 32 located on a slide 23. 4 shows a cross-sectional view, while FIG. 5 shows a plan view. 4 and 5 are not to be regarded as to scale to Figs. 2 and 3.
  • the biological object 32 is not planar on the slide 23.
  • the biological object 32 has a thickness which in principle makes it possible to sever the biological object with a single cut line.
  • the optimum focal point for the laser beam 31 for cutting the biological object 23 will depend on the location in the xy plane.
  • both a constant and / or predetermined distance from the focusing planes 35, 36, 37 and independent of the biological object 32 are variable and / or Object 32 dependent distance between the levels possible.
  • the choice of the respective focus plane for each section can be made in one embodiment via an autofocus function.
  • the choice of the focal plane for example, for tracing a cutting line as shown in Fig. 4 and 5 by an image analysis such as the aforementioned z-stacking done.
  • the microscope focus is set successively to a multiplicity of adjacent planes and an image is taken in each case with the image recording unit 31.
  • an overall image is then created from the respective "sharp", i.e. exactly focused parts of the individual images.
  • Embodiment of the invention stored and then used to irradiate the object with a laser beam as shown in Fig. 4 and 5.
  • the laser beam is focused in each case on the plane in which when recording the z-stack at the respective location, the greatest sharpness of the recording, ie the optimal focus was determined.
  • focusing would always be focused on the surface of the object.
  • a sharp image of the entire biological object on the slide can then first be displayed on the screen 22 of the computer 21 by means of the z-stack, whereupon the user sets, for example with the mouse 20, a desired cutting line.
  • This cutting line is then automatically swept programmatically with the laser beam 31, wherein the focusing of the laser on the planes (for example, planes 35, 36, 37 of FIG. 4) also takes place automatically program-controlled based on the data determined in the z-stacking.
  • Figs. 2-5 may also be combined, i.
  • a plurality of cutting lines can be executed one above the other, wherein in some or all of the cutting lines sections are focused on different planes.
  • the laser beam 31 is used for cutting out a region of interest from a biological object 30 or 32.
  • the laser beam can also be used for other purposes, with a focus of the laser beam on different planes perpendicular to the direction of the laser beam is used here as well.
  • fluorescent objects located in the biological object with the laser beam can be excited to fluoresce, and this fluorescence excitation can be recorded, for example, with the image acquisition unit 31 from FIG.
  • at least approximately to different points of the laser-facing surface of the biological object can be focused. It is also possible to focus on different planes within the biological object similar to FIG. 2.
  • the invention is not limited to the illustrated embodiments and structures, but a variety of modifications are possible.
  • an inverted microscope instead of using an inverted microscope, the use of an upright microscope is also conceivable, in which the laser beam is directed "from above" onto the biological object to be processed is merely illustrative, and other optical arrangements including, for example, lenses, mirrors, prisms, and the like are possible for this purpose, nor does the laser beam necessarily need to be perpendicular to a slide as in the embodiments of FIGS.
  • an oblique incidence is also possible
  • the focal planes can lie perpendicular to the laser beam or parallel to the respective microscope slide.

Landscapes

  • 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)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un procédé et un dispositif de traitement d'un objet biologique (30, 32). L'objet (32) est alors irradié avec un rayon laser (31) et le rayon laser est focalisé sur au moins deux plans focaux (35, 36, 37) différents. Pendant l'irradiation, le point focal du rayon laser se trouve au moins approximativement à l'intérieur de l'objet (32).
EP08774003A 2007-07-30 2008-07-16 Procédé et dispositif de traitement d'un objet biologique avec un rayon laser Ceased EP2181316A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200710035582 DE102007035582A1 (de) 2007-07-30 2007-07-30 Verfahren und Vorrichtung zum Bearbeiten eines biologischen Objekts mit Laserstrahlung
PCT/EP2008/005817 WO2009015775A1 (fr) 2007-07-30 2008-07-16 Procédé et dispositif de traitement d'un objet biologique avec un rayon laser

Publications (1)

Publication Number Publication Date
EP2181316A1 true EP2181316A1 (fr) 2010-05-05

Family

ID=39967391

Family Applications (1)

Application Number Title Priority Date Filing Date
EP08774003A Ceased EP2181316A1 (fr) 2007-07-30 2008-07-16 Procédé et dispositif de traitement d'un objet biologique avec un rayon laser

Country Status (3)

Country Link
EP (1) EP2181316A1 (fr)
DE (1) DE102007035582A1 (fr)
WO (1) WO2009015775A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011001474A1 (de) 2011-03-22 2012-09-27 Carl Zeiss Microimaging Gmbh Laser-Mikrodissektionsverfahren und Laser-Mikrodissektionsvorrichtung
AU2017343670A1 (en) 2016-10-13 2019-05-23 Sentek Pty Ltd Apparatus and method for anion detection and/or measurement

Family Cites Families (5)

* Cited by examiner, † Cited by third party
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
DE10015156A1 (de) * 2000-03-27 2001-10-18 P A L M Gmbh Verfahren und Vorrichtung zur Gewinnung eines biologischen Objekts aus einer biologischen Masse
DE10018255C2 (de) * 2000-04-13 2003-08-28 Leica Microsystems Laserschneid-Verfahren und Laserschneid-Vorrichtung zum Laserschneiden mit mikroskopischer Proben
DE10300091A1 (de) * 2003-01-04 2004-07-29 Lubatschowski, Holger, Dr. Mikrotom
DE102005028062C5 (de) 2005-06-16 2015-10-22 Leica Microsystems Cms Gmbh Laser-Mikrodissektionsverfahren und Vorrichtung zur Laser-Mikrodissektion

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2009015775A1 *

Also Published As

Publication number Publication date
DE102007035582A1 (de) 2009-02-05
WO2009015775A1 (fr) 2009-02-05

Similar Documents

Publication Publication Date Title
EP1745270B1 (fr) Procede de traitement d'une matiere par rayonnement laser et systeme de commande approprie
EP1893971B1 (fr) Procede et dispositif de microdissection laser
EP1583951B1 (fr) Microtome
DE10043506C1 (de) Verfahren und Vorrichtung zur Laser-Mikrodissektion
DE10018253A1 (de) Laser-Mikro-Dissektionsgerät
WO2007054161A1 (fr) Dispositif de manipulation d'echantillons
EP1985987B1 (fr) Procédé et dispositif de micro-dissection par laser
EP2689230B1 (fr) Procédé de microdissection au laser et dispositif de microdissection au laser
WO2014191383A1 (fr) Procédé de microdissection au laser et système de microdissection au laser
WO2009015775A1 (fr) Procédé et dispositif de traitement d'un objet biologique avec un rayon laser
WO2005033669A1 (fr) Procede de microdissection au laser
WO2008011944A1 (fr) Procédé et dispositif pour traiter des objets biologiques
WO2015128447A1 (fr) Système et procédé de microdissection laser
DE102014203656B4 (de) Lasermikrodissektionsverfahren und Verwendung eines Lasermikrodissektionssystems
DE102013209964B4 (de) Lasermikrodissektionssystem mit Benutzerinformationseinheit und Verfahren zur Lasermikrodissektion
DE102006009564B4 (de) Verfahren zur Bearbeitung einer Masse mittels eines Laserstrahls und entsprechende Vorrichtung
EP2499473A1 (fr) Procédé et dispositif pour examiner un système à laser
DE102019102852B3 (de) Verfahren zur Lasermikrodissektion, Lasermikrodissektionssystem und Computerprogramm
DE102020100587A1 (de) Verfahren zum Überprüfen eines Dissektiervorgangs in einem Laser-Mikrodissektionssystem und Mittel zu dessen Durchführung
DE102007030320B4 (de) Laser-Mikrodissektionsverfahren und Laser-Mikrodissektionssystem
WO2004040275A1 (fr) Microscopie par spectroscopie par claquage induit par eclair laser a l'aide d'impulsions laser ultracourtes

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20100226

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA MK RS

DAX Request for extension of the european patent (deleted)
RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: CARL ZEISS MICROSCOPY GMBH

17Q First examination report despatched

Effective date: 20151019

REG Reference to a national code

Ref country code: DE

Ref legal event code: R003

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20170129

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED