EP2181316A1

EP2181316A1 - Method and apparatus for treating a biological object with laser radiation

Info

Publication number: EP2181316A1
Application number: EP08774003A
Authority: EP
Inventors: Bernd SÄGMÜLLER
Original assignee: Carl Zeiss MicroImaging GmbH
Current assignee: Carl Zeiss Microscopy GmbH
Priority date: 2007-07-30
Filing date: 2008-07-16
Publication date: 2010-05-05
Also published as: WO2009015775A1; DE102007035582A1

Abstract

Provided are a method and an apparatus for treating a biological object (30, 32). Here, the object (32) is irradiated with a laser beam (31) and the laser beam is focused onto at least two different focal planes (35, 36, 37), wherein during the irradiation, the focus of the laser beam is located at least approximately inside the object (32).

Description

Method and device for processing a biological object with

laser radiation

The present invention relates to a method and an apparatus for processing a biological object by means of laser radiation.

A generic device is also known for example from EP 1 269 142 A of the applicant. In the device described there, for example, selected regions of biological objects can be cut out by computer-controlled means of a pulsed laser according to the principle of so-called laser microdissection. In this case, for example, a user can specify a cutting line by means of a computer, whereupon a computer-controlled cutting out of the desired region takes place from the biological object or the biological mass. Optionally, the areas thus cut out can then be catapulted by means of a laser pulse into a collecting container.

The device described in EP 1 269 142 A is particularly suitable for processing biological objects which lie planar on a slide and / or are not too thick, so that the laser beam can be focused in a simple manner on the plane of the object and it is sufficient to cut completely through the biological object, with the laser energy selected such that the cut-out area remains usable for subsequent examinations.

The device described in EP 1 269 142 A is also suitable for other types of processing of biological masses or objects. For example, fluorescent substances in a biological mass can be excited to fluorescence by means of a suitably focused laser beam. Again, the device is particularly suitable for the treatment of planar located on a slide biological objects of limited thickness.

It is therefore an object of the present invention to provide a method and a device with which thick and / or non-planar biological objects can be processed. This object is achieved by a method according to claim 1 and a device according to claim 15. The dependent claims define further embodiments of the method or the device.

A method according to the invention for processing a biological object, wherein the biological object is mounted on a carrier, comprises irradiating the biological object with a laser beam and automatically focusing the laser beam for irradiation on at least two different focal planes, wherein the focus of the laser is at least approximately within of the object, eg maximum 20 μm outside the object.

By automatically adjusting the laser focus to at least two different levels, it is also possible to process objects that are not thick or not planar on the support.

The at least two different levels may include a plurality of different levels at predetermined intervals. In other words, a given level grid can be used. In this case, the laser focus is at least approximately within the object when it is maximally on the plane of the plurality of planes immediately above or immediately below the object.

The method according to the invention is suitable for different types of biological objects, e.g. Tissue sections, cell cultures and the like.

In one embodiment, the irradiation of the object to cut out a region of interest from the object. In one exemplary embodiment, a closed cutting line with the laser is traversed on each at least two different planes, so that a stepwise cutting out of the region of interest can take place from a thicker object.

In another embodiment, additionally or alternatively, non-closed cutting lines are used on the at least two different planes. As a result, it is possible in particular to process objects that are not planar on the support. In order to determine, for example, for such non-closed cutting lines, where the laser beam is focused on which plane of the at least two planes, information from an imaging process such as the so-called z-stacking can be used. In z-stacking, an image of the object is taken, for example, by means of a microscope setup in different focal planes of a lens, a resulting image being composed of those parts of the individual images which are in focus. The planes for recording the individual images can comprise the at least two levels in an exemplary embodiment. In this case, the laser focus when irradiating the object can be set in each case to that level in which the best focus is present at the corresponding location in the imaging process.

The method of the invention can be used not only to excise regions of interest but, for example, to ablate (leach) a portion of the thickness of the object, to excite fluorescence in the biological object, and / or to bleach bleachable absorbers in the biological object. Other applications are possible.

A corresponding apparatus for processing an object comprises a laser, an optics for focusing the laser on an object located on the support and a focusing control, which is designed such that it moves the laser beam on at least two different planes parallel to the plane defined by the support focused during the irradiation of the object.

The invention will be explained in more detail with reference to the accompanying drawings with reference to preferred embodiments. Show it:

1 shows an embodiment of a device according to the invention,

2 shows a side view of a biological object for illustrating an embodiment of a method according to the invention,

3 is a plan view of the arrangement of Fig. 2,

4 shows a further side view of a biological object to illustrate a further embodiment of a method according to the invention, and 5 is a plan view of the arrangement of Fig. 4th

Embodiments of the invention will now be described with reference to FIGS. 1-5. 1 shows an exemplary embodiment of a device according to the invention for processing biological objects, in the form of a microscope system which is used, for example, for laser microdissection, i. can be used for cutting biological objects, for catapulting by means of laser radiation as well as generally for the manipulation of biological objects by means of laser radiation.

The system shown in FIG. 1 comprises a laser device 11 with a laser 18. The laser 18 may be, for example, a frequency tripled neodymium YAG laser. However, other types of lasers such as solid state lasers, gas lasers such as nitrogen lasers or dye lasers are possible. A laser beam emitted by the laser 18 is coupled through an optical system 15, 16 and mirror 17 into a microscope 13 and directed to a microscope objective 12 of the microscope 13. The optics 15, 16 comprises in the illustrated embodiment, a neutral density filter 15 for adjusting the intensity of the laser beam and lenses 16 for focusing the laser beam. In particular, this arrangement makes it possible to adjust the focusing of the laser beam both through the microscope objective 12 and through the lenses 16, so that the laser beam can be focused both together with the microscope 12 and independently thereof. The mirrors 17 can each be configured as conventional correspondingly coated mirrors or as beam splitters, for example in the form of prisms.

The microscope 13 shown in FIG. 1 is a so-called inverted microscope, in which the microscope 12 is arranged below a support table 14. On the support table 14 is then - for example, on an additional slide - an object to be processed, in particular a biological object or a biological mass arranged.

An image recording device 31 is provided in the microscope 13, for example a CCD camera with which an image of an object located on the support table 14 can be recorded via the microscope objective 12.

Furthermore, the exemplary embodiment illustrated in FIG. 1 has a collecting device 10 in which, for example, regions cut out of a biological object by means of the catapulting method mentioned in the introduction or in another way can be transported. In embodiments of the invention, which do not use such a catapulting method, for example, irradiate biological samples only for fluorescence measurements with lasers, the collecting device 10 can be omitted. Likewise, instead of an inverted microscope, as shown, an upright microscope can also be used in which the viewing of an object located on the support table and / or irradiation of the object with the laser beam occurs from above the support table.

The apparatus shown in Fig. 1 is controlled by a computer 21 having, in the illustrated embodiment, a data output screen 22 and a keyboard 19 and a mouse 20 for inputting data. It can also be provided more peripherals. The computer 21 controls in the embodiment in particular the laser device 11 with the lenses 16, the support table 14, which may be configured as a motorized xy-table, the neutral density filter 15 and optionally the catching device 10. In addition, via the computer 21 and the screen 22 of the camera 31 recorded images are evaluated and displayed. Furthermore, the computer 21 controls the microscope objective 12, which can be moved in the illustrated embodiment for focusing in the z-direction.

As can be seen from the coordinate system shown in FIG. 1, the plane of the support table 14 is assumed to be the xy plane, while the direction perpendicular to it is the z direction. Of course, these names are only for indication of direction and can be chosen differently.

In the illustrated embodiment, the laser beam, before it hits an object lying on the support table 14, thus runs in the z-direction.

The device illustrated in FIG. 1 can be cut out, for example, from a biological object located on the support table 14 by means of the laser beam emitted by the laser 18, an area of interest. For this purpose, for example, by means of the computer 21 to a captured with the camera 31 image of the biological object, a cutting line are set, which is then traversed automatically.

According to an embodiment, by using different focal planes of the objective 12 and / or the lenses 16, thicker biological objects can be cut in several steps. This will be exemplified below Referring to Figs. 2 and 3 explained. 2 shows a side view of a biological object 30 located on a slide 23. FIG. 3 shows a corresponding plan view. For example, slide 23 may be a laser beam 31 emitted by lasers 18, a transparent glass slide, or a membrane slide placed on support table 14 of FIG. In principle, it is also possible to arrange a biological object directly on the support table or, for example, in a container such as a petri dish, on the support table 14.

In the illustrated embodiment, the biological object 30 has a thickness that is so large that it can not readily be cut in one pass with the laser beam 31. In particular, in biological objects, the laser power is limited by the fact that although a cutting process to be performed, but the cut-out area and / or the non-cut portion of the biological

Object should be changed as little as possible. This results, for example, in a limitation of the section thickness of the order of magnitude of 20 .mu.m, which value may vary depending on the object to be processed and the laser used.

In the illustrated embodiment, an area is to be cut out, which is outlined by a section line 27, 28, 29 in Fig. 3. As already mentioned, such a cutting line can be entered by means of the computer 21 by a user.

In the exemplary embodiment illustrated in FIGS. 2 and 3, the cutting is carried out successively in different focal planes 24, 25, 26, which each have a distance Δz. The illustrated three levels 24, 25, 26 are to be understood merely as an example. Depending on the thickness of the biological object 30 and the step size Δz, more or less than three focal planes may be used. For example, in the case of a 100 μm-thick object, Δz can be 5 μm, so that 20 focal planes are used.

In each focal plane of the laser beam is in the embodiment of FIGS. 2 and 3 along a closed section line as shown in Fig. 3 moves. In particular, a cut line 27 is drawn in the focal plane 24, a cut line 28 is drawn in the focal plane 25 and a cut line 29 is drawn in the focal plane 26. In this way, the biological object 30 is cut through step by step. To set the focal planes, ie to focus the laser beam 31 on the respectively desired focal plane, the microscope objective 12 is moved in the z-direction in one exemplary embodiment. In another exemplary embodiment, the object carrier 23 is moved in the z-direction, for example by movement of the carrier table 14 from FIG. 1, in order to change the focal plane. In yet another embodiment, lenses within or outside the microscope objective, such as lenses 16 of FIG. 1, may be moved to adjust the focal plane. For tracing the cutting line in the xy plane, ie in the plane of the slide 23 and the planes 24, 25, 26, the object 30 is then moved relative to the laser beam 31, for example by an xy movement of the support table 14 of FIG. 1. However, it is also possible to leave the carrier table or the object in place and to move the laser beam 31, for example, by a mirror arrangement along the desired cutting line.

In an exemplary embodiment, the distances between the levels are constant. In this case, the distance may have a predetermined value, which is used independently of the object 30. However, the distance .DELTA.z can also be determined depending on the object used. In addition, the distances between the focal planes may also vary.

A further exemplary embodiment of the invention is explained below with reference to FIGS. 4 and 5. Similar to Figs. 2 and 3, Figs. 4 and 5 show a biological object 32 located on a slide 23. 4 shows a cross-sectional view, while FIG. 5 shows a plan view. 4 and 5 are not to be regarded as to scale to Figs. 2 and 3.

As shown in FIG. 4, the biological object 32 is not planar on the slide 23. In the example illustrated in FIGS. 4 and 5, the biological object 32 has a thickness which in principle makes it possible to sever the biological object with a single cut line. However, because the biological object 32 does not rest planar on the slide 23, the optimum focal point for the laser beam 31 for cutting the biological object 23 will depend on the location in the xy plane.

In the illustrated embodiment, therefore, when the cutting line is traversed, focus is focused on different focal planes 35, 36, 37, wherein the number of 3 focal planes is again to be understood as an example. In the illustrated example, for a portion 34 of the section line shown in Fig. 5 is focused on the plane 35, for a section 38 is focused on the plane 36, and for a section 33 is focused on a plane 37. The control of the focusing takes place, as in the example of FIGS. 2 and 3 computer-aided, in the apparatus of Fig. 1 by the computer 21st

Similar to the embodiment of FIGS. 2 and 3, also in the embodiment of FIGS. 4 and 5, both a constant and / or predetermined distance from the focusing planes 35, 36, 37 and independent of the biological object 32 are variable and / or Object 32 dependent distance between the levels possible.

The focusing of the laser and the departure of the cutting lines can be done in the embodiment of FIGS. 4 and 5 as already explained with reference to FIGS. 2 and 3.

The choice of the respective focus plane for each section can be made in one embodiment via an autofocus function. In another embodiment, the choice of the focal plane, for example, for tracing a cutting line as shown in Fig. 4 and 5 by an image analysis such as the aforementioned z-stacking done.

For this purpose, for example, in a device as in the exemplary embodiment of FIG. 1, the microscope focus is set successively to a multiplicity of adjacent planes and an image is taken in each case with the image recording unit 31. In conventional z-stacks, an overall image is then created from the respective "sharp", i.e. exactly focused parts of the individual images.

The information obtained in this method, in which lens setting in which part of the biological object is focused, is at a

Embodiment of the invention stored and then used to irradiate the object with a laser beam as shown in Fig. 4 and 5. For example, the laser beam is focused in each case on the plane in which when recording the z-stack at the respective location, the greatest sharpness of the recording, ie the optimal focus was determined. In such a case, as also shown in FIGS. 4 and 5, within the z-resolution of the z-resolution determined by the plane distance .DELTA.z, focusing would always be focused on the surface of the object. However, it is also possible, for example always a predetermined number of planes, for example a plane above or below this plane, which has the greatest sharpness in the z-stack frame, to focus the laser beam, if not focusing on the surface of the object, but slightly above or below desired is.

In such an embodiment, a sharp image of the entire biological object on the slide can then first be displayed on the screen 22 of the computer 21 by means of the z-stack, whereupon the user sets, for example with the mouse 20, a desired cutting line. This cutting line is then automatically swept programmatically with the laser beam 31, wherein the focusing of the laser on the planes (for example, planes 35, 36, 37 of FIG. 4) also takes place automatically program-controlled based on the data determined in the z-stacking.

Of course, the embodiments of Figs. 2-5 may also be combined, i. In the z-direction, a plurality of cutting lines can be executed one above the other, wherein in some or all of the cutting lines sections are focused on different planes.

In the embodiments explained so far with reference to FIGS. 2-5, the laser beam 31 is used for cutting out a region of interest from a biological object 30 or 32. In other embodiments, the laser beam can also be used for other purposes, with a focus of the laser beam on different planes perpendicular to the direction of the laser beam is used here as well. For example, fluorescent objects located in the biological object with the laser beam can be excited to fluoresce, and this fluorescence excitation can be recorded, for example, with the image acquisition unit 31 from FIG. In this case, according to the embodiment of FIGS. 4 and 5, at least approximately to different points of the laser-facing surface of the biological object can be focused. It is also possible to focus on different planes within the biological object similar to FIG. 2.

Even a so-called ablation process, in which some material is removed from the biological object, can be realized in this way. For example, according to FIG. 2, it would be possible to focus on the plane 24 in order to remove material in the lower part of the biological object facing the laser, and then it can For example, focusing on the plane 26 or an overlying plane to remove material in the upper part of the biological object, while the middle part remains unchanged. In the example of FIG. 4, a smoothing of the surface of the biological object would also be possible with such a method.

Other applications of the laser beam are also possible in this connection, for example activation or deactivation of parts, e.g. Nerve pathways, in a tissue sample or the so-called bleaching of elements such as bleachable absorbers or fluorescent elements.

The invention is not limited to the illustrated embodiments and structures, but a variety of modifications are possible. As already explained, instead of using an inverted microscope, the use of an upright microscope is also conceivable, in which the laser beam is directed "from above" onto the biological object to be processed is merely illustrative, and other optical arrangements including, for example, lenses, mirrors, prisms, and the like are possible for this purpose, nor does the laser beam necessarily need to be perpendicular to a slide as in the embodiments of FIGS. In principle, an oblique incidence is also possible Depending on the control, in this case the focal planes can lie perpendicular to the laser beam or parallel to the respective microscope slide.

Claims

PAT EN T AN SP RU CHE

Method for processing a biological object (30, 32), comprising: irradiating the biological object (30, 32) with a laser beam (31), and automatically focusing the laser beam (31) on at least two different focal planes (24, 25, 26, 35, 36, 37) during the irradiation of the biological object (30, 32), wherein during irradiation the focus of the laser beam lies at least approximately within the biological object (30, 32).

The method of claim 1, wherein the biological object is disposed on a support (14, 23), wherein the at least two different focal planes (24, 25, 26, 35, 36, 37) are parallel to a plane of the support.

3. The method of claim 1, wherein the at least two different focal planes (24, 25, 26, 35, 36, 37) are perpendicular to the direction in which the laser beam (31) is directed to the biological

Object (30, 32) hits.

4. The method of claim 1, wherein the at least two different focal planes (24, 25, 26, 35, 36, 37) have a predetermined distance (Δz) from each other.

5. The method of claim 1, wherein irradiating the biological object comprises cutting the biological object with the laser beam.

6. The method of claim 5, wherein in at least one of the at least two different focal planes (24, 25, 26) a closed cutting line (27, 28, 29) with the laser beam (31) is traversed.

7. The method according to claim 5 or 6, wherein at least one cutting line (33, 34, 38) is traveled in sections in different of the at least two different focal planes (35, 36, 37).

8. The method of claim 1, comprising: taking at least one image of the biological object, and

Selecting the at least two different focal planes in dependence on the at least one recorded image.

9. The method of claim 8, wherein in each case an image of the biological object (30, 32) is recorded in a plurality of different recording focal planes, and wherein the at least two different focal planes are selected in dependence on the recorded images.

10. The method of claim 9, wherein for each irradiated portion of the biological object (30, 32) that receiving focal plane is determined in which there is the best focus of each section to be irradiated, and, wherein the focal plane for irradiating the respective section in dependence is determined by the respective recording focus level with the best focus.

A method according to claim 9 or 10, wherein the at least two different focal planes are selected from the plurality of recording focal planes.

12. The method according to any one of claims 8 to 11, wherein the recording of the at least one image is carried out according to the z-stack method.

13. The method of claim 1, wherein irradiating the biological object with the laser beam comprises exciting fluorescent elements in the biological object.

14. The method according to any one of the preceding claims, wherein the irradiation of the object (30, 32) with the laser beam (31) comprises an ablation of a part of the biological object.

15. A device for processing a biological object, comprising: a laser (18) for generating a laser beam (31), optics (16, 17, 12) for focusing the laser beam (31) and for directing the laser beam (31) onto the biological object (30, 32), and a controller (19, 20, 21, 22) for controlling the focusing of the laser beam (31), wherein the controller (19, 20, 21, 22) is adapted to control the laser beam during an irradiation of the biological object (30, 32) focused on at least two different focal planes, wherein during irradiation, the focus of the laser beam is at least approximately within the biological object.

16. Apparatus according to claim 15, wherein the apparatus comprises a support (14, 23) for receiving the biological object (30, 32), the at least two focal planes (24, 25, 26; 35, 36, 37) being parallel to one another Level of the carrier (14, 23) are.

17. The apparatus of claim 15 or 16, wherein the at least two different focal planes (24, 25, 26, 35, 36, 37) are perpendicular to the direction in which the laser beam (31) hits the biological object.

18. Device according to one of claims 15-17, wherein the device comprises a microscope (13), wherein the optics comprises a lens (12) of the microscope.

19. An apparatus according to any one of claims 15-18, further comprising: an image pickup device (31) for picking up at least one image of the biological object (30, 32), wherein the controller (19, 20, 21, 22) is configured such that it selects the at least two focal planes as a function of an analysis of the at least one image of the biological object (30, 32).

20. Device according to one of claims 15-19, wherein the device for carrying out the method according to one of claims 1-15 is configured.