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WO2014191451A1 - Système de microdissection à laser comprenant un dispositif de visualisation et procédé de microdissection à laser - Google Patents

Système de microdissection à laser comprenant un dispositif de visualisation et procédé de microdissection à laser Download PDF

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Publication number
WO2014191451A1
WO2014191451A1 PCT/EP2014/061035 EP2014061035W WO2014191451A1 WO 2014191451 A1 WO2014191451 A1 WO 2014191451A1 EP 2014061035 W EP2014061035 W EP 2014061035W WO 2014191451 A1 WO2014191451 A1 WO 2014191451A1
Authority
WO
WIPO (PCT)
Prior art keywords
visualization
laser
laser microdissection
microdissection system
dissectate
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
PCT/EP2014/061035
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German (de)
English (en)
Inventor
Falk Schlaudraff
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.)
Leica Microsystems CMS GmbH
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Leica Microsystems CMS 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 Leica Microsystems CMS GmbH filed Critical Leica Microsystems CMS GmbH
Publication of WO2014191451A1 publication Critical patent/WO2014191451A1/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
    • 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
    • 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 laser microdissection system with a visualization device, a visualization device for such a laser microdissection system and a method for laser microdissection.
  • Laser microdissection has existed since the mid-1970s (see, eg, Isenberg, G. et al .: Cell surgery by laser micro-dissection: a preparative method, Journal of Microscopy, Vol. 107, 1976, pp. 19-24) and has been under continuous development since then ,
  • cells, tissue regions, etc. can be isolated from a tissue matrix and obtained as so-called dissectates.
  • a particular advantage of laser microdissection is the short contact of the tissue with the laser beam, through which the tissue is hardly changed. The specific recovery of the dissectates can take place in different ways (see, for example, Bancroft, J. D. and Gamble, M.: Theory and Practice of Histological Techniques, Elsevier Science, 2008, page 575, chapter "Laser Microdissection").
  • Ultraviolet laser beam is a segregated segregated either falls under the influence of gravity in a suitable Dissektatauffang effecter or catapulted against gravity in such a so-called laser
  • the dissectate can also be cut out of the object together with an adherent membrane.
  • a thermoplastic membrane which may also be connected to a reaction vessel, by means of a
  • the present invention is used in particular in the methods in which a dissectate is separated from an object and collected in a dissected collecting container.
  • the invention is suitable for contact-free collection systems for dissectates.
  • laser microdissection systems which have laser deflection or laser scanning devices which are set up to correspondingly displace the laser beam or its point of impact on the then stationary object are more advantageous.
  • Such laser microdissection systems which also offer particular advantages in the context of the present invention, are explained in detail below.
  • a particularly advantageous laser microdissection system comprising a laser scanning device with glass wedge plates is described in EP 1 276 586 B1.
  • Microscope lens used to focus the laser beam on the object and is therefore not available for imaging the Dissektatauffang feeling.
  • the object is located at the contactless
  • Working distance of the microscope objective for detecting the dissections in the dissektate collecting container is not sufficient, even a lens change may be required.
  • optimization work for example for
  • the present invention is based on a laser microdissection system, which is set up to separate a dissected object from an object to be dissected, for example a thin section on a slide, by means of a focused laser beam.
  • a laser microdissection system which is set up to separate a dissected object from an object to be dissected, for example a thin section on a slide, by means of a focused laser beam.
  • Laser microdissection systems focused a corresponding laser beam by means of a microscope objective on an object and the object thereby moved relative to the laser beam or vice versa.
  • a corresponding dissectate is collected in a dissektate catcher.
  • the laser microdissection system has means for receiving at least one corresponding one
  • the present invention thus relates in particular to those laser microdissection methods in which dissectates are separated out of an object and into a corresponding one
  • Dissectate collection containers fall or are transported without contact in these.
  • the present invention is a
  • Incident light device a microscope objective, a laser unit and a
  • Laser deflecting or -Scan has.
  • a beam path of a laser beam of the laser unit runs in this laser microdissection system through the incident light device, through the laser scanning device and through the laser scanning device Microscope lens and cuts an object plane of the microscope objective. It is therefore a laser microdissection system in which the laser beam is coupled into the incident light device and used by the respectively used
  • Microscope lens is focused on the object. That explained
  • Laser microdissection system corresponds in its basic structure, for example, that disclosed in EP 1276 586 Bl.
  • the intersection of the beam path of the laser beam with the object plane and thus the point of impact of the laser beam on the object can be moved by suitable deflection. This is done by deflecting the beam path of the laser beam in accordance with at least one set value of the mentioned laser adjustment means.
  • dissecting objects that are already prepared for microscopy. These may be, for example, thin sections which are separated out of a larger tissue block by means of a microtome.
  • 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 for the extraction of objects to be dissected but for their processing as well as for
  • microtome differs fundamentally in its operation from a laser microdissection system in that it produces cuts with as homogeneous a cutting thickness as possible. Microtomes are therefore designed to produce a large number of identical cuts with parallel cut surfaces, whereas laser microdissection systems for
  • Microtomes also do not include microscopes in which
  • Tissue block focused.
  • the object is mounted on one side in a laser microdissection system of the type discussed above, in bottom side upright systems, on a slide, and is processed by the slide with the laser beam from the other side thereof.
  • a laser microdissection system with a laser scanning device is used.
  • the microscope stage is in laser microdissection systems with a
  • Laserscan With respect to the xy direction (ie in the directions perpendicular to the optical axis of the microscope objective) when separating the dissecting, that is, during the Dissektiervorgangs stationary.
  • the laser scanning device is integrated in the incident light device. In contrast to laser microdissection systems with one during the
  • Dissecting process motorized microscope stage which is particularly high at high magnification lenses
  • laser microdissection systems using a laser scanning device are easier and less expensive to manufacture and have precision advantages.
  • the laser scanning device has two thick glass wedge plates which are inclined relative to an optical axis and can be rotated independently of one another about the optical axis, and which generate a beam deflection by means of their wedge angles. Due to the rotation of the glass wedge plates, the resulting deflection angle of the laser beam relative to the optical axis is variable.
  • the laser beam by the thickness and the inclination of the glass wedge plates a lateral
  • Such a laser scanning device is therefore particularly advantageous over other laser scanning devices such as mirror scanners, galvanometer scanners or stepper motor scanners because they do not have to be arranged in a plane conjugate to the objective pupil. Thus, no so-called pupil imaging is required in order to achieve that the deflected beam hits the objective pupil. In the case of microdissection with UV laser light, for example, a UV-capable pupil image would be required. Further Advantages of such a laser scanning device with wedge plates are mentioned, for example, in EP 1 276 586 B1.
  • the dissectate is in the laser microdissection system.
  • Dissektatauffang employs A visualization of dissections in a dissektate catcher.
  • a visualization of dissections in a dissektate catcher is also conventional
  • the invention provides for providing a visualization device which has one or more visualization cameras aligned or focusable on a collecting surface of the dissected collecting container and focused or focused independently of a focusing of the microscope objective.
  • the "collecting surface” is in particular the bottom of the
  • a completely independent solution for the visualization of dissectates in a dissektate collecting container is provided. Elaborate refocusing is completely eliminated.
  • the solution according to the invention can be used both in upright and in inverse laser microdissection systems. With the measures according to the invention, in particular the time required for the visualization of a dissektate collecting container or of the or in it
  • an object by a microscope objective or another
  • Dissecting be ensured that a corresponding dissecting has actually been collected in a Dissektatauffang practicer. Furthermore, it is possible, for example, the processing of the object by the laser beam stop immediately if a corresponding dissecting in
  • a corresponding lidded vessel is generally a tubular plastic vessel which may be tapered in the lower region and which has a vessel opening which is closable by the said lid
  • the lid which may serve as a dissected collecting vessel , Has a lid bottom as a collecting surface for the dissectate and on one side of the lid bottom at least one hollow cylinder which can be inserted through the vessel opening in a sealing seat on the vessel inner wall by folding the lid and thus
  • the dissectate can be carried into the lidded vessel.
  • An advantage of the solution according to the invention is in particular that by a suitable visualization camera (or multiple visualization cameras, which are directed together on the collecting surface of a Dissektatauffang actually uses the microscope objective as
  • a corresponding visualization device can advantageously have one or more visualization cameras, the opening side of the
  • Dissektatauffang observationsers are arranged.
  • Dissektatauffang tellers is usually aligned in the direction of the object.
  • Dissektatauffang falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang currently falls, the opening of the Dissektatauffang samples, as shown for example in Figures 2 and 3, located directly below the object.
  • the opening of the dissectate catcher is located above the object, and in such a case, a dissectate is carried by the laser beam into the dissektate catcher (up).
  • a "camera-side" of the Dissektatauffangites arranged visualization camera "looks" through the opening of the
  • the visualization camera or at least one of the several
  • Laser microdissection systems usually such that the object - on a suitable slide - between the lens and the
  • Dissektatauffang servinger is located.
  • a Visualization camera lens side of the object therefore "sees” the camera through the object and the opening of the Dissektatauffang actsers on its bottom or collecting surface.
  • the object must have sufficient transparency for this to a (sufficient) unobstructed view of the collection area of the
  • a corresponding transparency can also be created during the processing by the laser beam, which separates the dissectate from the object, a suitable opening is created, through which a detection of the collecting surface of the Dissektatauffang mattersers by means of the visualization camera is possible.
  • the visualization camera can
  • the visualization camera to the
  • a visualization camera can be installed in a microscope objective and / or by a suitable
  • Fastening device for example, a clamping socket and / or
  • the attachment to a microscope objective proves to be particularly simple, because through the microscope objective a defined position for the attachment of a corresponding
  • the visualization camera or at least one of the plurality of visualization cameras may prove to be advantageous, especially for reasons of space, the visualization camera or at least one of the plurality of visualization cameras
  • Visualization device on the bottom side of the Dissektatauffang practicers to order.
  • the visualization camera "looks" in this case through the bottom of the
  • Dissektatauffang servingers on the arranged in the Dissektatauffang servinger object It is understood that the bottom of the dissectate collecting container is transparent in such cases. The soil in these cases corresponds to the catchment area.
  • Visualization is detected, for example, can be realized by means of known microtiter plates in which dissektate collecting container are formed in the form of corresponding wells. Corresponding plates may be transparent, for example for spectrophotometric purposes, thereby allowing a dissecting of one to be considered
  • the visualization camera or at least one of the visualization cameras is designed with a laser protection device.
  • a laser protection device This can be realized for example in the form of appropriate filters.
  • At least one or more visualization cameras are designed to be movable in a corresponding laser microdissection system and / or can be controlled by control means.
  • control means for example, areas of a Dissektatauffang practicers, for example, sections of the
  • a deflection movement of the visualization camera or at least one of the plurality of visualization cameras can be effected when the dissectate collecting container is displaced. This makes it possible to cover the bottom of the dissected collecting container sufficiently by the camera and at the same time a movement of the
  • a corresponding laser microdissection system if between the object and the Dissektatauffang actually a light barrier or a sensor system is provided by means of which or a passing of a dissecting is detectable. This allows for a system response to a dissecting impact on the collection surface.
  • Visualization cameras for visualization may also include multiple dissectors and / or multiple Dissektatauffang practicern.
  • the visualization device likewise provided according to the invention for a corresponding laser microdissection system profits from the advantages explained above, to which reference is expressly made.
  • a visualization device is reversible at one
  • a method according to the invention for laser microdissection uses a corresponding laser microdissection system or a corresponding laser microdissection system
  • the dissectate may be particularly advantageously used during or immediately following a dissection process in the
  • Dissektatauffang servinger be visualisert be. As a result, it can be determined immediately when a corresponding dissection process is completed and its success is controlled directly.
  • Laser beam is used, the visualization camera (s) only during
  • Pulsphasen the pulsed laser beam is operated or will. This prevents the interference of interfering laser light on the
  • FIG. 1 shows a laser microdissection system that can be equipped with a visualization device according to an embodiment of the invention.
  • FIG. 2 shows details of a laser microdissection system according to FIG. 1 in a first embodiment in a schematic representation. shows details of a laser microdissection system according to Figure l in a second embodiment in a schematic representation.
  • FIG. 4 shows details of a laser microdissection system according to a first embodiment
  • FIG. 5 shows details of a laser microdissection system according to a second
  • Figure 1 is a laser microdissection system, which with a
  • the laser microdissection system 100 substantially corresponds to that disclosed in EP 1 276 586 B1, which is incorporated herein by reference.
  • the laser microdissection system 100 includes a microscope 10. In one
  • the Mikroskopfuß 11 of the microscope 10 may be provided only partially illustrated lighting device 12.
  • the laser microdissection system 100 is embodied as an upright system 100 in the illustrated example, but may also be designed in the same way as an inverse laser microdissection system.
  • the illumination device 12 may comprise, for example, 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 user input and / or user information unit 13 may be arranged, for example, as
  • Touchscreen can be formed, and on the user, for example, enter viewing and / or editing parameters and / or can read.
  • a drive knob 14 is provided, which may be designed to operate a coarse and a fine drive. By means of the drive knob 14, the height of a microscope stage 30 can be adjusted and thus an object 51, which is located on a
  • a condenser unit 20 is provided, which can be set up, for example, for phase contrast and / or for interference contrast illumination.
  • the arrangement of the illustrated components is known to be reversed, i. the lighting device 12 is at the top, the lens 41 at the bottom.
  • the laser microdissection system 100 has the above-mentioned and in the following figures illustrated in more detail Dissektatauffangs Skooth 50, by means of which dissecting the object 51 can be obtained.
  • the objective 41 is fastened to a lens revolver 40 in addition to other objectives 42.
  • a protective cover 15 may be provided to protect against laser radiation.
  • Auskoppel raised up to 60 °, coupled out and presented by means of an eyepiece pair 62 a user.
  • the illumination light for example, by 60 °
  • an eyepiece pair 62 a user.
  • Output coupling 61 designed to only a part of along the
  • a radiated observation light To couple out observation beam path a radiated observation light. Another portion passes into a digital image capture unit 63 and can be detected there, for example by means of a CCD chip or the like.
  • the laser microdissection system 100 has a laser unit 70 with a laser light source 75. A through the laser light source 75, in which it is
  • a UV laser light source may be provided
  • Laser beam b is deflected at a first deflection mirror 71 and a second deflection mirror 72 in a reflected-light unit, which is indicated here as a whole by 76, and focused onto the object 51 by the objective 41.
  • the location where the laser beam b is incident on the object 51 can be basically set in various 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.
  • electromechanical adjusting means may be provided which are controlled for example by a control unit 82 and their position by the
  • Control unit 82 can be detected.
  • the control unit 82 can also control any other functions of the laser microdissection system 100 and, in particular, provide an interface to an external control computer 81, which can be connected via corresponding connections 83.
  • control computer 81 and / or the drive unit 82 are set up to acquire images which can be obtained by means of visualization cameras 91 to 95 provided according to the invention (see FIGS. 4 and 5).
  • a corresponding adjusting device for the microscope stage 30 for the laser microdissection in the example shown is not necessary because the laser microdissection system 100 additionally has a laser scanning device 73.
  • the laser scanning device 73 By means of the laser scanning device 73, the laser beam b can also be deflected with respect to an optical axis c extending between the first deflection mirror 71 and the second deflection mirror 72.
  • the laser beam can therefore be at different positions on the second Deflection mirror 72 impinge, which may be formed, for example, as a dichromatic divider, and is thus focused at different positions on the object 51.
  • Laser scanning device 73 is shown in detail in EP 1 276 586 B1. It should be emphasized that different possibilities for deflecting a laser beam b or for positioning the object 51 relative to the laser beam b can be used. The invention is not limited to the illustrated example.
  • the laser scanning device 73 has two solid glass wedge plates 731, which are inclined relative to the optical axis c and are rotatable independently 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 may each be rotated by means of rotators 734.
  • the rotation means 734 can be rotated manually and / or by means of suitable electromechanical devices, for example by means of stepper motors, in a rotational movement and the above
  • the rotating devices 734 can over
  • FIG. 2 shows a cross section through the microscope stage 30 with the dissectant collecting device 50 mounted thereon in a plane spanned by the optical axis of the observation beam path a and the x direction (perpendicular to the plane of the paper of FIG. 1).
  • the microscope stage 30 has a fixed base plate 34, on which a in the y direction (that is perpendicular to the paper plane) movable plate 32 is arranged.
  • the movable plate 32 defines a table surface (without designation) of the microscope stage 30.
  • the movable plate 32 is opposite the fixed base plate 34 by means of a suitable device, for example under
  • bearing balls 33 movable.
  • adjusting device 31 For mutual displacement of the fixed base plate 34 and the movable plate 32 can already explained adjusting device 31 are used. If this is the case that the fixed base plate 34 "fixed”, this refers only to their position relative to the movable plate 32.
  • the "fixed” base plate 34 can in turn - in the x direction - against another plate of the microscope stage 30th shifted and - in the z-direction - be adjusted in height.
  • a dissektate collecting container 55 Arranged on the table surface of the microscope stage 30 is a dissektate collecting container 55, which optionally can be displaced by means of a suitable adjusting device (see FIG. However, the dissectate catcher 55 may also be manually moved in a free working space 54 of the dissektate catcher 50.
  • the dissektate collecting container 55 can also, as explained, be a lid of a known lidded vessel which can be clamped in the dissektate collecting device 50.
  • the sample collecting device 50 has a carrier element 53, on which a slide 52 can be placed.
  • the object 51 is attached to the underside of the slide 52.
  • the slide 52 is advantageously positioned so that the object 51 is completely in the free working space 54.
  • a height 541 of the free working space 54 may be adjustable by adjusting means and / or be defined by spacers (not shown).
  • the microscope objective 41 is on the optical axis of
  • Lens arrangement (without designation) an image of the object 51.
  • the object 51 is for this purpose in a focal plane of the microscope objective 41. Further, by the microscope objective 41 as explained to Figure 1 coupled laser beam b is focused on the object 51 to from the object 51st to cut out a corresponding dissectate.
  • the dissected collecting container 55 is arranged under the object 51 in such a manner that the dissectate due to the
  • Dissected 511 omitted.
  • Dissektatauffang practiceera 56 may be arranged, can be adapted to the respective requirements, for example, the number of dissectates 511 to be obtained.
  • FIG. 3 shows a corresponding section through a microscope stage 30 with a sample collecting device 50 arranged thereon according to another
  • Embodiment shown The sectional plane corresponds to that of Figure 2.
  • the sectional plane corresponds to that of Figure 2.
  • two holding elements 57 of which only the left holding member 57 with a
  • the holding elements 57 carry a
  • Contamination protection plate 58 the between the support member 53 and the Dissektatauffang employer 55, here together with others
  • Dissektatauffang preparationern 55 is attached to a Dissektatauffang experienceer( 56 is fixed.
  • the contamination protection plate 58 advantageously spans the entire table surface of the microscope stage 30 and thus limits the free working space 54 upwards.
  • Contamination protection plate 58 is provided with a cutout 581 about the optical axis of the observation beam path a.
  • a dissektate collecting container 55 of the Dissektatauffang etc. 56 for example, in each case a well of a known microtiter plate, can be arranged below the cutout 581.
  • Contamination protection plate 58 prevents dust or other particles from the ambient air from falling into dissektate receptacles 55. Furthermore, dissectates 511 of object 51 are prevented from being transformed into a "wrong" one.
  • Dissektatauffang fall when they are thrown out of the object 51 by the action of the laser beam b.
  • all but the respective desired dissectate collecting container 55, all other Dissektatauffang appearser 55 through the contamination protection plate 58th locked. This allows the use of the illustrated microtiter plates, which are a simple and cost-effective solution for collecting dissectates 511.
  • An adjustment device 59 may be connected to the dissektate receptacle carrier 56, wherein a corresponding engagement element 591 may be provided.
  • the adjusting device 59 can be operated manually, but preferably has a corresponding motor, by means of which the
  • the adjusting device 59 is for this purpose via a
  • connection 83 with a computer, for example, the explained control computer 81, connected.
  • the control computer 81 shifts over the
  • Adjustment device 59 the Dissektatauffang art or the Dissektatauffang noteder( 56 such that in each case the desired Dissektatauffang effecter 55 is below the cutout 581 and thus below the range of the object 51, which is processed by means of the laser beam b. After each laser microdissection process
  • Embodiments may be referred in particular to DE 100 18 251 C1, to which reference is hereby expressly made.
  • FIG. 4 schematically illustrates the visualization of a dissecting collecting container 55 according to a particularly preferred embodiment of the invention.
  • FIG. 4 corresponds to essential components of FIG. 2, wherein a corresponding sectional plane is also shown.
  • the height of the free working space 54 is increased in such a way (compare height 541) that in the free working space 54 a
  • Visualization camera 91 can be arranged. However, such an increase in the free working space 54 is not necessarily required when the visualization camera 91 is correspondingly small.
  • Visualization camera 91 is positioned so that it can preferably detect the entire bottom of the dissectate collection container 55 (see observation cone 911), which here corresponds to the collection surface. If this is not possible, for example for reasons of space, a corresponding visualization camera 91 may also only partially detect the bottom of the dissecting collecting container 55, wherein preferably a complete image of the bottom of the dissecting collecting container 55 is obtained by joining individual images (so-called stitching).
  • stitching Several visualization cameras 91 for detecting partial areas can also be provided.
  • the visualization camera 91 may be designed to be movable, wherein corresponding (not shown) actuating means may be provided. However, the visualization camera 91 is at least adjustable in order to allow the most complete detection of the bottom of the Dissektatauffang disposers 55.
  • a corresponding line 84 is provided for forwarding the images obtained by means of the visualization camera 91.
  • the line 84 the images of the visualization camera 91 in any form, the
  • Visualization camera 91 provides, will be forwarded.
  • the forwarding in the form of analog and / or digital signals and / or by means of a suitable transmission protocol. Examples of suitable
  • Transmission protocols or formats are USB and / or mJPEG. In order to allow the smallest possible formation of the visualization camera 91, this can only be provided with minimal image processing means. Alternatively, the visualization camera 91 may perform rendering (e.g., sharpening) to some extent.
  • the dissected collecting container 55 for example, on a corresponding Dissektatauffang practiceer Diego 56 is movable relative to the visualization camera 91.
  • a Dissektatauffang servinger 55 or its bottom, can therefore be moved into the detection cone 911 of the visualization camera 91.
  • the visualization camera 91 can therefore also be fixed with respect to the optical axis of the observation beam path a and / or correspondingly adjustable be.
  • the visualization camera 91 may also be designed to be movable in relation to the optical axis of the observation beam path a, so that it can independently scan the bottom of the dissektate collecting container 55.
  • FIG. 4 a corresponding Inverssystem is shown. This corresponds essentially to that of the main view, but the arrangement of the microscope objective 41, the slide 52 with the object 51 attached thereto, the dissektate collecting container 55 and the
  • Visualization camera 91 is rotated 180 ° with respect to the horizontal. In such Inverssystemen a dissectate is ejected from the object 51 by the action of the laser beam b. The bottom of the
  • Dissektatauffang observationsers 55 here is preferably provided with an adhesive coating, so that a dissectate at the bottom of the
  • Dissektatauffang servingers 55 may adhere.
  • a further embodiment of the present invention is schematically illustrated in FIG. 5, which is based on the embodiment shown in FIG. As in FIG. 3, a dissektate collecting container holder 56 is displaceable by means of an adjusting device 59 or an engagement element 591 attached thereto.
  • FIG. 5 shows different embodiments of the present invention
  • Visualization cameras here labeled 92-95, differ. Again, the solution shown in Figure 4 can be used, i. a visualization camera corresponding to the visualization camera 91 can be mounted above the dissecting collection container 55. Conversely, all arrangements shown in FIG. 5 can also be used in FIG.
  • Dissecting Tray Carrier 56 (with transparent bottom).
  • transparent bottom For example, as the dissecting receptacle holder, transparent
  • Microtiter plates are used, the bottom of which can be detected from below by means of the visualization camera 92. Since the visualization camera 92 in the example shown directly on the optical axis of
  • Avoid laser beam b It can also be provided to operate the visualization camera 92 with a time offset to the laser beam b. For example, as
  • Laser beam b uses a pulsed laser beam b, the
  • Visualization camera 92 activated only during pulse pauses of the laser beam b.
  • the visualization camera 92 is advantageously positioned and / or dimensioned so that it does not interfere with transmitted light illumination. For this purpose, but also to avoid damage to a visualization camera 92 by the laser beam b, can also be provided, these off-axis the optical axis of the observation beam path a, and thus outside a target position of
  • the visualization camera 92 can also be fixedly arranged relative to the optical axis of the observation beam path a and / or correspondingly adjustable and / or adjustable.
  • the visualization camera 92 and also the further visualization cameras 93 to 95 of FIG. 5 are connected to the control computer 81 or the control unit 82 via lines 84 (shown only in the case of the visualization camera 92).
  • the arrangement of the visualization camera 93 corresponds in principle to the arrangement of the visualization camera 91 from FIG. 4.
  • the representation of the detection cone of the visualization camera 93 has been dispensed with.
  • Visualization camera 93 is in the area of the cutout 581 of
  • the visualization camera 93 can, in order to ensure an adjustability of the Dissektatauffang practiceer(s 56, be designed adjustable in height, preferably in an adjustment step of the Dissektatauffang employer(s 56, for example by the
  • Adjustment device 59 an automatic lifting of the visualization camera 93 (ie, an "evasive movement") is effected
  • the walls of the Dissektatauffang effecter 55 of the Dissektatauffangfläzentertowns 56 may be provided with suitable recesses, the free mobility of the
  • Dissektatauffang afternooner(s 56 in x and y direction allow. It can also be provided, the visualization camera 93 in the illustrated
  • Contamination protection plate 58 may be designed to be transparent and / or have transparent areas formed.
  • Visualization cameras 91 to 95 can trigger.
  • the light barrier and / or the sensor system can likewise be connected to a control unit 82 and / or the control computer 83. It can also be provided
  • a visualization camera 94 may also be mounted in or on a microscope objective 41.
  • Visualization camera 94 can be firmly integrated into the microscope objective 41 and / or reversibly attached to it, for example by means of a suitable slip-on sleeve 941.
  • the visualization camera 94 makes it possible, with sufficient transparency of the object 51, the bottom of the
  • Dissektatauffang the bottom of the disseparate collecting container 55 can still be detected, namely when, by the laser beam b and by the cutting out of the corresponding dissecting, a sufficiently large window is created in the object 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)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un système de microdissection à laser (100) qui comporte un microscope (10) doté d'un système d'éclairage (76) pour la focalisation d'un rayon laser (b) par un objectif de microscope (41) du microscope (10) sur un objet à disséquer (51) et des moyens de séparation d'un produit de dissection (511) de l'objet à disséquer (51) au moyen du rayon laser focalisé (b). Le système de microdissection à laser (100) comporte en outre des moyens de réception d'un récipient de réception de produit de dissection (55) pour recevoir le produit de dissection (511) et des moyens de visualisation du produit de dissection (511) dans le récipient de réception du produit de dissection (55). Les moyens de visualisation du produit de dissection (511) comportent dans le récipient de réception du produit de réception (55) une ou plusieurs caméras de visualisation (91-95) dirigées ou pouvant être dirigées sur une surface de réception du récipient de réception du produit de dissection (55) et focalisées ou pouvant être focalisées indépendamment d'une focalisation de l'objectif du microscope (41) sur le produit de dissection dans le récipient de réception du produit de dissection (55). L'invention concerne également un procédé correspondant.
PCT/EP2014/061035 2013-05-28 2014-05-28 Système de microdissection à laser comprenant un dispositif de visualisation et procédé de microdissection à laser Ceased WO2014191451A1 (fr)

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DE102013209881.2 2013-05-28
DE102013209881.2A DE102013209881A1 (de) 2013-05-28 2013-05-28 Lasermikrodissektionssystem mit Visualisierungseinrichtung, Visualisierungseinrichtung für Lasermikrodissektionssystem und Verfahren zur Lasermikrodissektion

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CN111929883A (zh) * 2020-09-10 2020-11-13 广西医科大学第一附属医院 用于小型实验动物离体标本显微解剖的辅助调整定位装置

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CN111929883A (zh) * 2020-09-10 2020-11-13 广西医科大学第一附属医院 用于小型实验动物离体标本显微解剖的辅助调整定位装置

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