WO2014191451A1

WO2014191451A1 - Laser microdissection system comprising a visualization apparatus and method for laser microdissection

Info

Publication number: WO2014191451A1
Application number: PCT/EP2014/061035
Authority: WO
Inventors: Falk Schlaudraff
Original assignee: Leica Microsystems Cms Gmbh
Priority date: 2013-05-28
Filing date: 2014-05-28
Publication date: 2014-12-04
Also published as: DE102013209881A1

Abstract

A laser microdissection system (100) is proposed, which comprises a microscope (10) with an epi-illumination apparatus (76) for focussing a laser beam (b) through a microscope objective (41) of the microscope (10) onto an object (51) to be dissected and means for separating a dissected specimen (511) from the object (51) to be dissected by means of the focussed laser beam (b), wherein the laser microdissection system (100) furthermore comprises means for holding a dissected specimen collection container (55) for collecting the dissected specimen (511) and means for visualizing the dissected specimen (511) in the dissected specimen collection container (55). The means for visualizing the dissected specimen (511) in the dissected specimen collection container (55) comprise one or more visualization cameras (91-95) which are or can be aligned on a collection surface of the dissected specimen collection container (55) and are or can be focussed on the dissected specimen in the dissected specimen collection container (55) independently of a focussing of the microscope objective (41). A corresponding method is likewise subject matter of the invention.

Description

Laser microdissection system with visualization device and method for laser microdissection Description

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.

State of the art

Method for processing microscopic samples by the so-called

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 , In the case of laser microdissection, 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").

For example, from an object using an infrared or

Ultraviolet laser beam is a segregated segregated either falls under the influence of gravity in a suitable Dissektatauffangbehälter or catapulted against gravity in such a so-called laser

Pressure Catapulting). The dissectate can also be cut out of the object together with an adherent membrane. In the so-called laser capture microdissection, however, a thermoplastic membrane, which may also be connected to a reaction vessel, by means of a

heated corresponding laser beam. The membrane merges with the desired area of the object and can be removed in a subsequent step. Another alternative is to attach the dissectate by means of the laser beam to a lid of a Dissektatauffangbehälters. In known inverse microscope systems for laser microdissection, catapulted dissected-up can also be attached to the bottom of a dissektate collection container provided with an adhesive coating.

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. In particular, the invention is suitable for contact-free collection systems for dissectates.

Known microscope systems for laser microdissection, as they are known for example from WO 98/14816 AI, have a Auflichteinrichtung, in whose beam path a laser beam is coupled. The laser beam is focused by the respectively used microscope objective on the object, which rests on a motor-automatically movable microscope stage. A cutting line is created by moving the microscope stage during cutting to move the object relative to the fixed laser beam. However, this has the disadvantage, among other things, that the object can not be viewed well during the generation of the cutting line, since it moves continuously or stepwise in the field of view.

Therefore, 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.

In both cases, so both in laser microdissection systems, in which the microscope stage is moved, as well as in laser microdissection systems, the have a laser scanning, is usually worked with pulsed lasers, each hole being generated in the object by each laser pulse, for example. A cutting line is created by a juxtaposition of such holes, possibly with overlap. The laser microdissection is usually performed with fully or semi-automatic microscope systems, with a monitor for visualization of the object and of corresponding cutting lines is provided. Even with the earliest systems, as described, for example, in the article by Isenberg et al. Although a mechanical microscope is used, the visualization is also done here via a "television camera" in conjunction with a monitor ("TV").

Due to the principle, in the non-contact laser microdissection methods, ie those in which the sepectates fall out of the object into a dissektate collecting container after removal from the object or are conveyed without contact, no simultaneous display of the dissected fractions is possible during the dissection process:

As explained, in the laser microdissection, the respectively used

Microscope lens used to focus the laser beam on the object and is therefore not available for imaging the Dissektatauffangbehälters. The object is located at the contactless

Laser microdissection procedure during a dissection process

necessarily in the focal plane of the microscope objective, but the dissected areas outside the focus plane. Therefore, in conventional systems, the success of the dissection process can only be verified after this. This requires a refocusing. In cases where the free

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. In particular, in optimization work, for example for

Determining the laser power required for dissection is this

extremely expensive. There is therefore a need for improved laser microdissection systems which in particular allow a simultaneous display of an object and / or the dissectates obtained therefrom during the dissection process.

Disclosure of the invention

Against this background, the present invention proposes

Laser microdissection system with a visualization device, a

Visualization device for such a laser microdissection system and a method for laser microdissection with the features of the independent claims before. Preferred embodiments are subject of the dependent claims and the following description.

Advantages of the invention

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. As already explained, in known

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

Dissektatauffangbehälters on. 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. In other words, the present invention is a

Laser microdissection system with a microscope consisting of a

Incident light device, a microscope objective, a laser unit and a

Laser deflecting or -Scaneinrichtung 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.

By the laser scanning device, 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.

Is in the context of this application of a "point of impact" of the laser beam on an object or its "target point" the speech, this is about

Positions that are directly from the intersection of the beam path of the

Laser beam with the object plane result when an object is placed in this. These terms can therefore be used synonymously.

To avoid misunderstandings it should be emphasized at this point that the laser microdissection system used in the context of the invention also belongs to

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. Such a tissue block may be, for example, a fixed organ or a biopsy of a corresponding organ. The laser microdissection system according to the invention therefore does not serve for the extraction of objects to be dissected but for their processing as well as for

Isolation of certain areas thereof. It is understood that the invention may also be used with other dissecting objects which are not obtained by means of a microtome, for example with smears, macerates, etc. Microtomes are used exclusively in the preparation of microscopic objects. Microtomes can also have lasers for this purpose. The sections obtained by means of a microtome are applied to a microscope slide as mentioned above, possibly attached there, stained, etc. Only then are these available for use in the laser microdissection system according to the invention. Among other things, a 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

Dissecting segregates according to object-dependent criteria, for example, according to visual criteria, are established. The skilled person would therefore not in technical microtomes used solutions

Transfer laser microdissection systems. Microtomes also do not include microscopes in which

Observation beam a laser beam is coupled. The laser beam in microtomes will therefore never be transmitted through a microscope objective, which is also used for viewing, onto a processed object, e.g. one

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.

Furthermore, in the context of the present invention, advantageously a laser microdissection system with a laser scanning device is used. The microscope stage is in laser microdissection systems with a

Laserscaneinrichtung 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 (scanning table), which is particularly high at high magnification lenses

To have positioning accuracy to enable precise cuts, laser microdissection systems using a laser scanning device are easier and less expensive to manufacture and have precision advantages.

Furthermore, a viewer in laser microdissection systems with a microscope stage which is moved by a motor during the dissecting process, moves the image during the dissecting process. This is especially annoying when viewed with a slow camera and a monitor. The

Monitor image blurs in this case and shows jerky changes. This disadvantage shows laser microdissection systems with a

Laser scanning device, however, not because the microscope stage is fixed.

In a particularly advantageous embodiment, 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. At the output of the laser scanning device, the laser beam by the thickness and the inclination of the glass wedge plates a lateral

Beam offset from the optical axis and hits the center of the lens pupil of the microscope objective for all deflection angle.

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.

In the laser microdissection system, the dissectate is in the

Dissektatauffangbehälter visualizable. A visualization of dissections in a dissektate catcher is also conventional

Laser microdissection systems possible, wherein the microscope objective itself is used for visualization. This requires a refocusing with the mentioned disadvantages.

In contrast, 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

Dissektatauffangbehälters. Generally a "catchment area" is one

Dissektatauffangters the area on which a dissectate after the

Slicing out of the object hits.

Thus, according to the invention, 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

arranged dissectates reduced. In particular, an object (by a microscope objective or another

Visualization device) and the dissectate are visualized in the dissektate catcher. As a result, for example, immediately after the

Dissecting be ensured that a corresponding dissecting has actually been collected in a Dissektatauffangbehälter. Furthermore, it is possible, for example, the processing of the object by the laser beam stop immediately if a corresponding dissecting in

Diss ect t catcher hit.

If in the context of the present invention the term "dissektate collecting container" is concerned, it can also be, in particular, a lid of a known lidded vessel, as is customarily used in the laboratory for receiving small quantities of liquid A corresponding lidded vessel is described for example in EP 1 317 961 In a particular embodiment, 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

Closing the lidded vessel, 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 Dissektatauffangbehälters), the entire collecting surface of Dissektatauffangbehälters can be mapped in one step. When using the microscope objective as

Visualization device, however, often the collecting area of a corresponding Dissektatauffangbehälters, in particular the mentioned lid of the lid vessels, for example, from 0.5 ml vessels, laboriously searched (scanned), because the microscope objective, if necessary, a considerable

Magnification has. A dissectate can thus be found much faster by means of the solution according to the invention.

If, for example, for documentation purposes, images are stored electronically, this proves to be particularly advantageous because a Composing a corresponding image from several fields (by so-called stitching) is not required.

The microscope objective used by the inventively embodied visualization constantly in the optimal focus position for

Processing and / or observation of the object remain. A refocusing or a lens change is not required.

A corresponding visualization device can advantageously have one or more visualization cameras, the opening side of the

Dissektatauffangbehälters are arranged. The opening side of the

Dissektatauffangbehälters is usually aligned in the direction of the object. In systems in which a dissecting by gravity in the

Dissektatauffangbehälter falls, the opening of the Dissektatauffangbehälters, as shown for example in Figures 2 and 3, located directly below the object. In inverse systems (see view A in Figure 2), 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 Dissektatauffangbehälters arranged visualization camera "looks" through the opening of the

Dissektatauffangbehälters on the bottom or collecting surface. Such an arrangement proves to be particularly favorable, because between the

Visualization camera and the collecting area of Dissektatauffangbehälters no disturbing structures are present, which could hinder observation of the collecting surface of the Dissektatauffangbehälters.

In certain cases, especially for reasons of space, can also

be provided, the visualization camera or at least one of the several

Visualization cameras lens side of the object to arrange. The arrangement of the objective, the object and the Dissektatauffangbehälters is known

Laser microdissection systems usually such that the object - on a suitable slide - between the lens and the

Dissektatauffangbehälter is located. In an arrangement of a Visualization camera lens side of the object therefore "sees" the camera through the object and the opening of the Dissektatauffangbehälters 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

Dissektatauffangbehälter to allow. 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 Dissektatauffangbehälters by means of the visualization camera is possible. In an objective-side arrangement of the visualization camera can

be provided in particular, the visualization camera to the

Attach microscope objective or this to a corresponding

Make microscope objective attachable by appropriate means for coupling are provided. A visualization camera can be installed in a microscope objective and / or by a suitable

Fastening device, for example, a clamping socket and / or

appropriate sleeves are attached to the microscope objective. This provides an additional viewing opportunity for the object and the

Dissektatauffangbehälter created. 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

Visualization camera is provided.

In contrast to the cases explained above, however, it 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 Dissektatauffangbehälters to order. The visualization camera "looks" in this case through the bottom of the

Dissektatauffangbehälters on the arranged in the Dissektatauffangbehälter 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. A corresponding arrangement by which a Dissektatauffangbehälter thus arranged with a "condenser side"

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

Bottom side of the Dissektatauffangbehälters off.

Advantageously, in a laser microdissection system according to the invention, the visualization camera or at least one of the visualization cameras is designed with a laser protection device. This can be realized for example in the form of appropriate filters.

Advantageously, 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. As a result, for example, areas of a Dissektatauffangbehälters, for example, sections of the

Floor area, to be scanned.

In certain cases, in particular when the dissectate collecting container is displaceable by an adjusting device, 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

Restrict the Dissektatauffangbehälter in the least possible extent.

Particularly advantageous is a corresponding laser microdissection system, if between the object and the Dissektatauffangbehälter 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.

Advantageously, in this case the light barrier or the sensor system with the visualization camera or with at least one of the plurality Visualization cameras coupled so that it can be triggered via the photocell or the sensor system.

It should be stressed that even in cases where previously of "one"

Visualization camera or "a" Dissektat or Dissektatauffangbehälter the question is, the invention also the use of several

Visualization cameras for visualization may also include multiple dissectors and / or multiple Dissektatauffangbehältern.

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. In particular, such a visualization device is reversible at one

Dissektatauffangseinrichtung the laser microdissection system attachable.

A method according to the invention for laser microdissection uses a corresponding laser microdissection system or a corresponding laser microdissection system

Visualiser. Also to the features of the invention

Method is therefore made to the advantages and features explained above.

In such a method, the dissectate may be particularly advantageously used during or immediately following a dissection process in the

Dissektatauffangbehälter be visualisert be. As a result, it can be determined immediately when a corresponding dissection process is completed and its success is controlled directly.

Advantageously, in laser microdissection methods a pulsed

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

Visualization camera (s).

figure description

The present invention and embodiments of the invention will be explained below with reference to the accompanying drawings. 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

Embodiment of the invention in a schematic representation. FIG. 5 shows details of a laser microdissection system according to a second

Embodiment of the invention in a schematic representation.

In the figures, corresponding elements are identical with each other

Reference numerals indicated and not explained repeatedly.

In Figure 1 is a laser microdissection system, which with a

Visualization device according to an embodiment of the invention can be equipped, shown schematically and designated 100 in total. 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

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. At the microscope head 11, for example, 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.

Further, 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

Slide (without designation) is located on a Dissektatauffangeinrichtung 50, for example, applied to the slide section are brought into a focus plane of a lens 41. For transparent illumination of the object and for setting suitable contrast or observation methods, a condenser unit 20 is provided, which can be set up, for example, for phase contrast and / or for interference contrast illumination. In inverse laser microdissection systems, 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 Dissektatauffangseinrichtung 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. To protect against laser radiation, a protective cover 15 may be provided.

From the object 51 outgoing observation light runs along a

Observation beam path a. In a tube unit 60 with suitable

Auskoppeleinrichtungen 61, at least a portion of the illumination light, for example, by 60 °, coupled out and presented by means of an eyepiece pair 62 a user. In the example shown is the

Output coupling 61 designed to only a part of along the

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

For example, 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.

In the illustrated laser microdissection system 100, the location where the laser beam b is incident on the object 51 can be basically set in various ways. On the one hand, 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. In addition to the adjusting device 31 and 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.

In particular, the 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).

For the laser microdissection, 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. 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. A corresponding diversion by means of a

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.

In 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. For this purpose, 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

Drive gears 733. The rotating devices 734 can over

Position transmitters 735 (shown here only on the right rotation device 734). A position detected thereby can be transmitted to the control unit 80. 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

Use of bearing balls 33, movable. 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.

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

Observation beam path a (here, as shown in Figure 1, dash-dotted lines) facing the slide 52 and generates a corresponding

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

Gravity falls in these. A dissectate 511 is in the

Dissektatauffangbehälter 55 shown. In the following figures was off For clarity, the representation of a corresponding

Dissected 511 omitted. The number of Dissektatauffangbehälter, as shown in the following Figure 3, also on a common

Dissektatauffangbehälterträger 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. In the example shown are on the fixed base plate 34, two holding elements 57, of which only the left holding member 57 with a

Reference numeral is attached attached. The holding elements 57 carry a

Contamination protection plate 58, the between the support member 53 and the Dissektatauffangbehälter 55, here together with others

Dissektatauffangbehältern 55 is attached to a Dissektatauffangbehälterträger 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. The

Contamination protection plate 58 is provided with a cutout 581 about the optical axis of the observation beam path a.

In each case a dissektate collecting container 55 of the Dissektatauffangbehälterträgers 56, for example, in each case a well of a known microtiter plate, can be arranged below the cutout 581. By the

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.

Dissektatauffangbehälter 55 fall when they are thrown out of the object 51 by the action of the laser beam b. In other words, all but the respective desired dissectate collecting container 55, all other Dissektatauffangbehälter 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

Engagement element 591 and the attached Dissektatauffangbehälterträger 56 can be moved. The adjusting device 59 is for this purpose via a

corresponding connection 83 with a computer, for example, the explained control computer 81, connected. The control computer 81 shifts over the

Adjustment device 59 the Dissektatauffangbehälterträger 56 such that in each case the desired Dissektatauffangbehälter 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

is completed, the corresponding dissectate 511 falls into the dissectate catcher 55 by gravity. The control computer 81 will then, if necessary, position another or empty dissektate catcher 55 accordingly to catch one or more new dissectates 511. For further embodiments of the illustrated in Figures 2 and 3

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.

In the example 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. The

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). 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 Dissektatauffangbehälters 55. For forwarding the images obtained by means of the visualization camera 91, a corresponding line 84 is provided. By means of the line 84, the images of the visualization camera 91 in any form, the

Visualization camera 91 provides, will be forwarded. For example, 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 Dissektatauffangbehälterträger 56 is movable relative to the visualization camera 91. In each case a Dissektatauffangbehälter 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. However, 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.

As explained, the present invention is suitable for both upright and inverse systems. In view A of Figure 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

Dissektatauffangbehälters 55 here is preferably provided with an adhesive coating, so that a dissectate at the bottom of the

Dissektatauffangbehälters 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

Invention, characterized by the positioning of appropriate

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.

It may be provided, a visualization camera 92 below a

Dissektatauffangbehälters 55 or a corresponding

Dissecting Tray Carrier 56 (with 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

Observation beam path a and thus arranged on the optical axis of the laser beam b, it is advantageously provided with suitable protection devices that damage the visualization camera 92 through the

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

Position laser beam b. In this case, the camera - according to the visualization camera 91 of Figure 4 - obliquely to the bottom of the

Dissektatauffangbehälters 55 directed. In the example shown in FIG. 1, 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. The

Visualization camera 93 is in the area of the cutout 581 of

Contamination protection plate 58 arranged. The visualization camera 93 can, in order to ensure an adjustability of the Dissektatauffangbehälterträgers 56, be designed adjustable in height, preferably in an adjustment step of the Dissektatauffangbehälterträgers 56, for example by the

Adjustment device 59, an automatic lifting of the visualization camera 93 (ie, an "evasive movement") is effected Alternatively, the walls of the Dissektatauffangbehälter 55 of the Dissektatauffangfläzenterträgers 56 may be provided with suitable recesses, the free mobility of the

Dissektatauffangbehälterträgers 56 in x and y direction allow. It can also be provided, the visualization camera 93 in the illustrated

Orientation above the contamination protection plate 58 to order. The

Contamination protection plate 58 may be designed to be transparent and / or have transparent areas formed.

At the location of the visualization camera 93, it is also possible to arrange a light barrier and / or a suitable sensor system which detect passage of a dissectate 511 and on this basis one of the

Visualization cameras 91 to 95 can trigger. For this purpose, 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

store corresponding image only when the light barrier and / or the sensor system triggers.

According to another embodiment, a visualization camera 94 may also be mounted in or on a microscope objective 41. The

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

Dissektatauffangbehälter 55 through the slide 52 and the object 51 to detect therethrough. If there is no sufficient transparency of the object 51, 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.

By means of one or more further visualization cameras 95 further Dissektatauffangbehälter 55 of the Dissektatauffangbehälterträgers 56 can be detected, which, for example, allows a parallel evaluation.

Claims

claims

A laser microdissection system (100) comprising a microscope (10) having incident light means (76) for focusing a laser beam (b) through

Microscope objective (41) of the microscope (10) on an object to be dissected (51) and means for separating a Dissektats (511) from the zu

dissecting object (51) by means of the focused laser beam (b), wherein the laser microdissection system (100) further comprises means for receiving a dissektate collection container (55) for collecting the dissectate (511) and means for visualizing the dissectate (511) in the dissektate collection container (55 ), characterized in that the means for visualizing the

Dissectats (511) in the Dissektatauffangbehälter (55) one or more on a collecting surface of the Dissektatauffangbehälters (55) aligned or alignable and independent of a focus of the microscope objective (41) on the dissectate in the Dissektatauffangbehälter (55) focused or focusable visualization cameras (91- 95).

2. Laser microdissection system (100) according to claim 1, wherein the

Visualization camera (91-95) or at least one of the several

Visualization cameras (91-95) opening side of the Dissektatauffangbehälters (55) is arranged.

3. laser microdissection system (100) according to claim 2, wherein the

Visualization camera (91-95) or at least one of the several

Visualization cameras (91-95) lens is arranged on the side of the object (51).

4. laser microdissection system (100) according to claim 3, wherein the

Visualization camera (91-95) or at least one of the several

Visualization cameras (91-95) attached to the microscope objective (41) or attachable.

5. laser microdissection system (100) according to claim 1, wherein the

Visualization camera (91-95) or at least one of the several Visualization cameras (91-95) arranged on the bottom side of the Dissektatauffangbehälters (55) and the bottom of the Dissektatauffangbehälters (55) is transparent.

6. laser microdissection system (100) according to one of the preceding

Claims in which the visualization camera (91-95) or at least one of the plurality of visualization cameras (91-95) comprises a laser protection device.

7. laser microdissection system (100) according to one of the preceding

Claims in which the visualization camera (91-95) or at least one of the plurality of visualization cameras (91-95) is designed to be movable and / or in the control means (81, 82) for driving the visualization camera (91-95) or at least one of the plurality Visualization cameras (91-95) are provided.

8. laser microdissection system (100) according to claim 7, wherein a

Adjustment device (59) for moving the Dissektatauffangbehälters (55) and means for effecting an evasive movement of the visualization camera (91-95) or at least one of the plurality of visualization cameras (91-95) when moving the Dissektatauffangbehälters (55) are provided.

9. laser microdissection system (100) according to one of the preceding

Claims, wherein between the object (51) and the Dissektatauffangbehälter (55) a light barrier or a sensor system for detecting a

passing dissecting (511) is provided.

10. laser microdissection system (100) according to claim 9, wherein the

Photoelectric sensor or the sensor system with the visualization camera (91-95) or with at least one of the plurality of visualization cameras (91-95) is coupled.

11. Laser microdissection system (100) according to one of the preceding

Claims in which the means for separating the dissectate (511) from the object to be dissected (51) by means of the laser beam (b) focused on the object (51) by the microscope objective (41) comprises a laser scanning device (73) in the incident light device (76 ) for variably deflecting the laser beam (b).

12. Laser microdissection system (100) according to claim 11, wherein the

Laser scanning device (73) inclined against an optical axis and independently of each other about the optical axis rotatable glass wedge plates (731).

13. Visualization device for use in one

A laser microdissection system (100) according to any one of the preceding claims, which is one or more focused or focusable on the collection surface of the dissectate collection container (55) and focused on a dissectate (511) in the dissektate collection container (55) regardless of focusing of the microscope objective (41) Visualization cameras (91-95).

14. A visualization device according to claim 13, comprising means for reversible attachment to a Dissektatauffangseinrichtung (50).

15. A method for laser microdissection, wherein a

A laser microdissection system (100) according to any one of claims 1 to 12, wherein a dissectate (511) is visualized in a dissector collection container (55) by visualization means by one or more aligned or orientable and independent of a collection surface of the dissector collection container (55) a focus of the

Microscope lens (41) focused or focusable visualization cameras (91-95) are used.

16. The method of claim 15, wherein the dissectate (511) in the

Dissectate collection container (55) during or immediately after one

Dissection process is visualized.

17. The method of claim 15 or 16, wherein a pulsed laser beam (b) is used and the visualization camera (91-95) or at least one of the plurality of visualization cameras (91-95) only during pulse pauses of the pulsed laser beam (b) is operated.