DE102007035582A1 - Method and device for processing a biological object with laser radiation - Google Patents

Abstract

A method and apparatus for processing a biological object (30, 32) is provided. In this case, the object (32) is irradiated with a laser beam (31) and the laser beam focused on at least two different focal planes (35, 36, 37), wherein during the irradiation, the focus of the laser beam at least approximately within the object (32).

Description

The 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 for example from the EP 1 269 142 A the applicant known. 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 in the EP 1 269 142 A described device is particularly suitable for the processing of biological objects which rest planar on a slide and / or are not too thick, so that the laser beam can be focused on the plane of the object in a simple manner and is sufficient to cut through the biological object completely , wherein the laser energy chosen so that the cut-out area remains usable for subsequent investigations.

The in the EP 1 269 142 A The device described 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, a method and to provide a device with which thick and / or not planar lying biological objects are machinable.

These The object is achieved by a method according to claim 1 or a device according to claim 15. The dependent Claims define further embodiments the method or the device.

One Inventive method for processing a biological object, wherein the biological object on a support attached, includes irradiating the biological object with a laser beam and the automatic focusing of the laser beam for irradiating at least two different focal planes, wherein the focus of the laser at least approximately within the object lies, z. B. a maximum of 20 microns outside of the object.

By the automatic adjustment of the laser focus to at least two Different levels can not be fat or not planar objects lying on the support are processed.

The At least two different levels can be a variety 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, if he is maximally on the immediate level above or immediately below the object the variety of levels is located.

The inventive method is suitable for various types of biological objects, e.g. B. tissue sections, Cell cultures and the like.

at In one embodiment, the irradiation of the object takes place for cutting out a region of interest from the object. In one embodiment, each is at least two on each different levels a closed cutting line with the laser traversed, allowing a gradual cutting out of the area of interest can be made from a thicker object.

at Another embodiment will be additional or alternatively not closed cut lines on the at least used two different levels. In particular, this can not planar objects resting on the support are processed.

Around for example, for such non-closed cutting lines to determine where the laser beam is at which level of the at least Focusing two levels can provide information from an imaging Method such as the so-called z-stacking used become. In z-stacking, for example, by means of a microscope assembly in different focal planes of a lens, an image of the object taken, with a resulting image of those parts the frames is composed, which focuses sharply are. The layers for taking pictures can be included an embodiment, the at least two levels include. In this case, the laser focus when irradiating the object respectively be set to the level in which at the corresponding Place in the imaging process is the best focus.

The inventive method can not only for Cut out areas of interest are used, but for example, for ablating (ablating) a portion of the thickness of Object, for exciting fluorescence in the biological object and / or bleaching bleachable absorbers in the biological Object. Other applications are possible.

A corresponding device for processing an object comprises a Laser, an optic for focusing the laser on an on-carrier Object and a focusing control, which designed in such a way is that they parallel the laser beam on at least two different levels to the plane defined by the support during irradiation focused on the object.

The Invention will be described below with reference to the attached Drawing with reference to preferred embodiments closer explained. Show it:

1 an embodiment of a device according to the invention,

2 a side view of a biological object to illustrate an embodiment of a method according to the invention,

3 a plan view of the arrangement of 2 .

4 a further side view of a biological object to illustrate a further embodiment of a method according to the invention, and

5 a plan view of the arrangement of 4 ,

In the following, embodiments of the invention with reference to 1 - 5 described. 1 shows an embodiment of an inventive device for processing biological objects, in the form of a microscope system, which can be used for example for laser microdissection, ie for cutting biological objects, for catapulting by laser radiation and also generally for the manipulation of biological objects by means of laser radiation.

This in 1 The system shown comprises a laser device 11 with a laser 18 , The laser 18 For example, it may be 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. One from the laser 18 emitted laser beam is through an optic 15 . 16 and mirrors 17 in a microscope 13 coupled and to a microscope lens 12 of the microscope 13 directed. The optics 15 . 16 includes 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, by this arrangement, an adjustment of the focusing of the laser beam by both the microscope objective 12 as well as through the lenses 16 possible so that the laser beam both together with the microscope 12 as well as independently of this can be focused. The mirror 17 can each be configured as a conventional corresponding coated mirror or as a beam splitter, for example in the form of prisms.

This in 1 illustrated microscope 13 is a so-called inverted microscope in which the microscope 12 below a carrier table 14 is arranged. 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.

In the microscope 13 is an image capture device 31 provided, for example, a CCD camera, with which on the microscope objective 12 a picture of one on the support table 14 located object can be recorded.

Furthermore, the in 1 illustrated embodiment, a collecting device 10 in which, for example, areas cut out of a biological object can be conveyed by means of the catapulting method mentioned in the introduction or in some other way. In embodiments of the invention which do not use such a catapulting method, for example irradiating biological samples only for fluorescence measurements with lasers, the collecting device can 10 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.

In the 1 The device shown is by a computer 21 controlled, which in the illustrated embodiment, a screen 22 for data output as well as a keyboard 19 and a mouse 20 to enter 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 carrier table 14 , which as motori xy-table can be configured, the neutral density filter 15 and optionally the collecting device 10 , In addition, over the computer 21 and the screen 22 from the camera 31 recorded images are evaluated and displayed. Furthermore, the computer controls 21 the microscope objective 12 which can be moved in the illustrated embodiment for focusing in the z direction.

Like the one in 1 To take the coordinate system shown, the plane of the support table 14 assumed as 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 passes before moving to one on the support table 14 lying object hits, thus in z-direction.

The in 1 shown device may, for example, from one on the support table 14 located biological object by means of the laser 18 emitted laser beam are cut out a region of interest. For this purpose, for example, by means of the computer 21 on one with the camera 31 recorded image of the biological object, a cutting line are set, which is then automatically traversed.

According to an embodiment, by using different focal planes of the objective 12 and / or the lenses 16 thicker biological objects are cut in several steps. This will be exemplified below with reference to 2 and 3 explained. It shows 2 a side view of one on a slide 23 located biological object 30 , 3 shows a corresponding plan view. slides 23 For example, one may be for one of the lasers 18 emitted laser beam 31 be transparent glass slide or a membrane slide, which on the support table 14 out 1 is arranged. In principle, it is also possible to have a biological object directly on the support table or, for example, in a container such as a petri dish located on the support table 14 to arrange.

In the illustrated embodiment, the biological object 30 a thickness which is so great that it is not readily in one pass with the laser beam 31 can be cut. In particular, in the case of biological objects, the laser power is limited by the fact that, although a cutting process is to be carried out, the cut-out region and / or the non-cut-out region 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, which by a cutting line 27 . 28 . 29 in 3 outlined. As already mentioned, such a cutting line can be done by means of the computer 21 be entered by a user.

At the in 2 and 3 illustrated embodiment, the cutting is successively in different focal planes 24 . 25 . 26 performed, each having a distance .DELTA.z. The illustrated three levels 24 . 25 . 26 are to be understood only as an example. Depending on the thickness of the biological object 30 and the pitch .DELTA.z can be used more or less than three focal planes. 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 in the embodiment of 2 and 3 along a closed cutting line as in 3 shown moves. in particular, in the focal plane 24 a cutting line 27 pulled, in the focal plane 25 a cutting line 28 pulled and in the focal plane 26 a cutting line 29 drawn. In this way, the biological object becomes 30 gradually cut through.

For adjusting the focal planes, ie for focusing the laser beam 31 to the respective desired focal plane, in one embodiment, the microscope objective 12 moved in z-direction. In another embodiment, the slide becomes 23 , For example, by movement of the support table 14 out 1 , moved to change the focal plane in the z direction. In yet another embodiment, lenses may be inside or outside the microscope objective, such as lenses 16 out 1 , are moved to adjust the focal plane. To move the cutting line in the xy plane, ie in the plane of the slide 23 or the levels 24 . 25 . 26 , then becomes the object 30 relative to the laser beam 31 moved, for example by an xy movement of the support table 14 out 1 , However, it is also possible to leave the support table or the object in place and the laser beam 31 for example, to move by a mirror assembly along the desired cutting line.

In one embodiment, the distances between the levels are constant. In this case, the distance may be a predetermined value which, regardless of the object 30 is used. 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.

Another embodiment of the invention will be described below with reference to FIG 4 and 5 explained. Similar to in 2 and 3 demonstrate 4 and 5 a biological object 32 which is on a microscope slide 23 is located. 4 shows a cross-sectional view while 5 shows a top view. The 4 and 5 are not as true to the scale 2 and 3 to watch.

As in 4 represented is the biological object 32 not planar on the slide 23 on. At the in 4 and 5 Example shown has the biological object 32 a thickness which basically allows the biological object to be severed with a single cut line. Because the biological object 32 but not planar on the slide 23 is the optimal focal point for the laser beam 31 for cutting the biological object 23 dependent on location in the xy plane.

In the illustrated embodiment, therefore, when traversing the cutting line to different focal planes 35 . 36 . 37 focussed, again taking the number of 3 focal planes as an example.

In the example shown is for a section 34 the in 5 shown cutting line on the plane 35 focused, for a section 38 gets to the level 36 focused, and for a section 33 gets to a level 37 focused. The control of the focusing takes place as well as in the example of 2 and 3 computer-aided, in the device of 1 through the computer 21 ,

Similar to the embodiment of the 2 and 3 is also in the embodiment of 4 and 5 both a constant and / or independent of the biological object 32 predetermined distance between the focus planes 35 . 36 . 37 as well as a variable and / or object 32 dependent distance between the levels possible.

The focusing of the laser and the departure of the cutting lines can in the embodiment of the 4 and 5 as already referring to 2 and 3 explained.

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 in 4 and 5 represented by an image analysis such as the z-stacking mentioned above.

For this purpose, for example, in a device as in the embodiment of 1 the microscope focus is successively set to a plurality of adjacent planes and one image each with the image pickup unit 31 added. In conventional z-stacks, an overall image is then created from the respectively "sharp", ie exactly focused parts of the individual images.

The information obtained in this method, in which objective adjustment is focused in which part of the biological object, is stored in one embodiment of the invention and then irradiated with a laser beam as in 4 and 5 used. For example, the laser beam is focused in each case on the plane in which when taking the z-stack at the respective location, the greatest focus of the recording, ie the optimal focus was determined. In such a case, as well as in 4 and 5 is shown, within the z-resolution of the focusing determined by the plane distance .DELTA.z always focused on the surface of the object. However, it is also possible, for example, always a predetermined number of levels, for example, a plane above or below this level, which has the greatest sharpness in the z-stack frames to focus the laser beam, if no focus on the surface of the object but something above or below is desired.

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

Of course, the embodiments of the 2 - 5 can also be combined, ie it can be performed in the z-direction several cutting lines one above the other, wherein in a part or in all cutting lines is focused in sections on different levels.

When referring to 2 - 5 previously explained embodiments, the laser beam 31 for cutting out a region of interest from a biological object 30 respectively. 32 used. 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 excited, for example, with the image recording unit 31 out 1 be recorded. It can according to the embodiment of 4 and 5 be focused at least approximately on different points of the laser-facing surface of the biological object. It is equally possible to different levels within the biological object similar to in 2 to focus.

Even a so-called ablation process, in which some material is removed from the biological object, can be realized in this way. For example, it could be appropriate 2 to the level 24 be focused to ablate material in the lower, the laser-facing part of the biological object, and then, for example, to the plane 26 or an overlying plane to ablate material in the upper part of the biological object, while the middle part remains unchanged. In the example of 4 With such a method it would also be possible to smooth the surface of the biological object.

Also Other applications of the laser beam are in this context possible, for example, an activation or deactivation of parts, eg. As neural pathways, in a tissue sample or so called bleaching of elements such as bleachable absorbers or Fluorescent members.

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 conceivable in which the laser beam is directed "from above" onto the biological object to be processed 1 illustrated optical structure for directing the laser beam into the lens 12 is merely illustrative, and other optical arrangements including, for example, lenses, mirrors, prisms and the like are possible for this purpose. Also, the laser beam does not necessarily have to be as in the embodiments of FIG 1 - 5 perpendicular to a slide or a corresponding support table meet, but it is basically an oblique incidence possible. In this case, depending on the control, the focal planes may be perpendicular to the laser beam or parallel to the respective slide.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - EP 1269142 A [0002, 0003, 0004]

Claims (20)

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 irradiation of the biological object ( 30 . 32 ), wherein during irradiation the focus of the laser beam is at least approximately within the biological object ( 30 . 32 ) lies. The method of claim 1, wherein the biological object is supported 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 carrier. Method according to 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 ) on the biological object ( 30 . 32 ) meets. Method according to claim 1, wherein the at least two different focal planes ( 24 . 25 . 26 ; 35 . 36 . 37 ) have a predetermined distance (Δz) to each other. Method according to one of the preceding claims, wherein the irradiation of the biological object ( 30 . 32 ) a cutting of the biological object ( 30 . 32 ) with the laser beam ( 31 ). Method according to claim 5, wherein in at least one of the at least two different focal planes ( 24 . 25 . 26 ) a closed section line ( 27 . 28 . 29 ) with the laser beam ( 31 ) is traversed. Method according to claim 5 or 6, wherein at least one cutting line ( 33 . 34 . 38 ) in sections in different of the at least two different focal planes ( 35 . 36 . 37 ) is traversed. Method according to one of the preceding claims, comprising: receiving at least one image of the biological object ( 30 . 32 ), and selecting the at least two different focal planes in response to the at least one captured image. A method according to claim 8, wherein in each case an image of the biological object (A) in a plurality of different receiving focal planes ( 30 . 32 ), and wherein the at least two different focal planes are selected depending on the captured images. Method according to claim 9, wherein for each section of the biological object to be irradiated ( 30 . 32 ) is determined that receiving focal plane, in which the best focus of the respective section to be irradiated is present, and, wherein the focal plane for irradiating the respective section in dependence on the respective recording focus plane is determined with the best focus. The method of claim 9 or 10, wherein the at least two different focal planes from the variety of recording focal planes to be selected. Method according to one of claims 8 to 11, wherein the taking of the at least one image according to the z-stack method he follows. Method according to one of the preceding claims, wherein the irradiation of the biological object ( 30 . 32 ) with the laser beam exciting fluorescent elements in the biological object ( 30 . 32 ). Method according to 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. Apparatus for processing a biological object, comprising: a laser ( 18 ) for generating a laser beam ( 31 ), an optic ( 16 . 17 . 12 ) for focusing the laser beam ( 31 ) and for directing the laser beam ( 31 ) on the biological object ( 30 . 32 ), and a controller ( 19 . 20 . 21 . 22 ) for controlling the focusing of the laser beam ( 31 ), whereby the controller ( 19 . 20 . 21 . 22 ) in such a way that it irradiates the laser beam during 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. Device according to claim 15, wherein the device comprises a carrier ( 14 . 23 ) for receiving the biological object ( 30 . 32 ), wherein the at least two focal planes ( 24 . 25 . 26 ; 35 . 36 . 37 ) parallel to a plane of the carrier ( 14 . 23 ) are. Apparatus according to 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. Device according to one of Claims 15-17, the device being a microscope ( 13 ), the optics being a lens ( 12 ) of the microscope. The apparatus of any one of claims 15-18, further comprising: an image capture device ( 31 ) for capturing at least one image of the biological object ( 30 . 32 ), whereby the controller ( 19 . 20 . 21 . 22 ) is configured such that it determines the at least two focal planes as a function of an analysis of the at least one image of the biological object ( 30 . 32 ) selects. Device according to one of claims 15-19, wherein the apparatus for performing the method according to one of claims 1-15 is configured.