DE19616216A1 - Laser beam process and assembly separates individual cells from tissue mass - Google Patents

Abstract

Laser beam which process and assembly separates and sorts a range of biological objects such as tissue cells comprises: (a) a microscope (2) for the observation of objects such as tissue samples resting on a substrate (4); (b) a focused laser beam (13) directed at \- 1 biological item(s) (10) under preparation, where the laser source and substrate may be moved relative to each other, and (c) a capture vessel (18, 20) located behind the substrate which intercepts the selected objects which are ejected (14) from the sample by the laser beam.

Description

The invention relates to a method and an apparatus for sorting and separating for individual biological objects. The objects are here a fixed planar carrier arranged side by side. With this Methods can be made from a very large number of objects (e.g. 10⁵-10⁹) individual objects are spatially separated and separated. The Separation of clustered cells as a whole is also possible. The method can also be used to separate specific cells Tissue sections can be used. Requirement for this Sorting process is the prior recognition and selection of the concerned Objects based on specific properties (e.g. through coloring, Fluorescent labeling or by radioactive labeling). Under "biological objects" are used in the context of the present application especially living or fixed biological cells or cell components Roger that.  

Objects can be used to separate individual biological objects optical methods, such as optical tweezers (Optical Tweezer) in one moving aqueous solution (K. Schütze, A. Clement-Sengewald, Nature, 667 (Vol. 368) 1994). This method is due to the low power transmission limited to objects that can move freely in the solution. That I the sorted as the unsorted objects are in the same solution, separate cultivation can only be achieved with additional effort. For a separate cultivation, these cells have to be mixed with another one Method such as B. can be separated or aspirated with microcapillaries. Adherent cells can grow with fine needles that cross over Micromanipulators are moved, separated. Here, the Cells touched directly and could therefore be mechanically loaded. Both Methods are relatively time-consuming, so that they are not used Processing a variety of objects are suitable.

For separating individual cells from a large number (<10⁶) in one Liquid dispersed, biological objects suitable separation or Sorters are commercially available. While at the fluorescence activated cell sorting (FACS = Fluorescence activated Cell Sorter) electrostatic principles for spatial separation come, the magnetically activated cell sorter (MACS = Magnetic activated cell sorter) with magnetic forces. Here are the cells but not side by side on a planar support. Moreover have Both methods have the disadvantage that some objects are only restricted (FACS) or not separated at all (MACS).

The methods presented cannot contain cells from a cell cluster such as a tissue or from histological tissue preparations.  

Furthermore, under the name "Ablative Photodecomposition" are procedures known in which with pulsed UV lasers, in particular with excimer Lasers, a targeted material removal with polymers takes place. This procedure can be viewed in the broadest sense as an etching process. On similar process, but with a continuously operated UV Laser is used is described in U.S. Patent 5,211,805. This Process is intended for industrial processing of technical Polymers and for biomedical treatment of biological Tissue. This is related to a sorting principle, that with Laser beams the unwanted on a carrier biological objects with high radiation doses destroyed during the selected (desired) objects remain (US 4,624,915). This Process is relatively complex to make single objects from large ones To select populations.

The object underlying the invention is spatial Separation of individual biological objects that are side by side on one solid planar supports such. B. a glass slide are. The process of secretion should be as short as possible (<10 s) and non-contact, e.g. B. feasible in separate, sterile chambers. The process should also be very reliable and therefore simple be automatable. At the same time, the survivability of the biological objects usually remain guaranteed; d. H. the biological Objects should not be damaged or impaired by the separation process will.

In the patent application 196 03 996.7 from 05.02.1996, this task solved in that an area of a carrier film on which the selected biological object, cut out with a laser beam and passed through  a laser-induced transport process to an immediately above or catcher substrate arranged below the carrier film is transferred. The solution to this earlier proposal is therefore that biological objects in a small, preferably circular environment cut out together with the carrier film by a laser beam and together with the cut-out part of the carrier film on one in the Be flung near the catcher. Apparently this is happening due to the light pressure of the laser beam, since the separated parts of the Carrier film together with the biological object on it always in the direction of Laser beam are accelerated.

To further improve the method according to the aforementioned patent application 196 03 996.7, a new and improved solution to the problem underlying the invention is provided in the present case in that the light pressure of the laser beam is now to be used to directly remove the desired particle or biological object from the surface of the object to catapult away solid planar support (object holder or petri dish) and catch it in a suitable vessel, ie the separation of a biological object is also possible without simultaneously removing or cutting out the area of the carrier film carrying the biological object. According to the present innovation or improvement, this takes place in the following manner, which is to be explained with reference to the attached FIGS. 1 to 3:

When using an upright microscope 2 according to FIG. 1, a suitable slide 4 (thickness approximately 170 μm) is placed in reverse on the holder 6 of a microscope stage 8 specially developed for laser micromanipulation, ie the biological object 10 is located on the underside of the slide 4 . The object is e.g. B. histological tissue sections of a few microns thickness, or adhering chromosome or DNA preparations.

The microscope stage 8 is designed for a displacement in 2 (for x / y movement) or 3 axes (for x / y / z movement) and with a holding device 6 for z. B. slide 4 or Petri dishes. The table 8 is therefore motorized via suitable drives, which are computer-controlled in a known manner, for. B. can be moved using a computer mouse (or joystick). The hybrid stepper motors of the axis drives work with high precision. The smallest step is 20 nm, the maximum travel path of the microscope table 8 can be up to several centimeters (e.g. 10 cm). The precision for retrieving stored points is <400 nm. Speeds for moving the microscope table from almost zero to 25 mm / second can be selected. With a so-called frame grabber, the current microscope image recorded via a video camera is put on a monitor and can be processed with computer functions, e.g. B. command functions, control characters, control marks, etc., are graphically superimposed (video overlay).

Only thin slides (approx. 170 µm thick) or petri dishes with thin (approx. 25 µm), gas-permeable film (so-called Petriperm dishes) are suitable for laser micromanipulation. Since high demands are placed on precise sample holding and sample guiding for laser micromanipulation in the nanometer range, the holding holder 6 has been specially configured: With the thin slides 4 there is a danger that B. with the use of oil immersion lenses slightly bend and thus no good focus can be achieved. To avoid this, the slide 4 must rest on at least 3 sides of the holders, whereby only the two narrow sides of the slide 4 can be clamped with a spring clip. Another necessity, especially for laser microscopy, is the exact adjustability of the sample holder (slide 4 or holder 6 ). It must be ensured that the sample is always at the same distance from the tip of the objective 12 , specifically in the entire travel range of the carrier (approximately 5-10 cm).

First, suitable biological objects 10 are optically selected with a smaller magnification. As soon as all interesting objects are saved in the computer, you switch to a higher magnifying lens. The beam offset caused by changing the lens (chromatic aberration) is compensated for by a correction function on a saved value, which is then automatically transferred to all saved points.

The computer now moves to the first object 10 . The microscope image recorded by a video camera is displayed on the computer monitor not shown in the figures. A marker on the monitor shows the position of the laser beam focus. The microscope stage is either moved by hand (controlled by mouse or joystick) or moves automatically in a circular or spiral manner around the selected object 10 , controlled by a computer program according to a predetermined pattern. The "marker" on the monitor can be viewed as a "pen" with which the contour of the desired biological object is traced. If the laser fires at a pulse frequency of approximately 15-20 Hz at the same time, all material that is in the firing line in the area of the "marker" is removed or destroyed. The extremely focused laser beam "draws" a fine line of approximately 500 nm in width around the desired object 10 and thus separates it from its surroundings. In the case of cells in a histological section, this procedure detaches the desired cell from the cell cluster and loosens it from the substrate in the form of the slide 4 . By spiraling around the selected target cell mentioned above, the lasered area around the cell is enlarged.

The laser used here is a pulsed, compact nitrogen laser with high beam quality (wavelength: 337 nm, Pulse duration: 3 nsec (nanoseconds), pulse frequency: from 10 to 20 Hertz). Other lasers are also conceivable, provided that the laser wavelength used is biological material is not adversely affected. The laser beam itself or its source preferably remains immobile. However, the laser can also in x / y direction with a radius of approximately 100 µm relative to Object level are moved, d. H. ultimately it is only important that laser beam and the object plane (microscope stage) are moved relative to one another.

The object prepared in this way can now be catapulted into a sample vessel automatically with a further, targeted laser shot, faster and more securely than in the prior art (for example with a needle) and above all without contact, that is to say completely sterile, if necessary (trajectory 14 ).

For this purpose, it is necessary that a second holder 16 (e.g. to clamp a collecting vessel 18 or a microtiter plate) is moved under the first holder 6 via two motorized and computer-controlled axes so that the object 10 , which has been freed by laser, is positioned exactly above the collecting vessel 18 is placed. Here, too, high precision of the motor movement is a prerequisite for a clean collection of the desired objects. A single, targeted laser shot (possibly slightly defocused) throws the selected biological object 10 or the cell in the beam direction (trajectory 14 ) into the collecting vessel 18 . Then a new object can be cut out and the whole process is repeated.

To speed up the collection process, all desired objects can first be prepared with the laser. The collecting vessel 18 is then moved under the microscope stage. The microscope stage moves to the stored, laser microdissected objects one after the other. One shot at a time conveys the individual objects in each case into fresh (new) receptacles 18 , FIG. 1, which are transported further in coordination with the movement of the microscope stage 8 .

In the case of an inverted microscope ( FIG. 2), an adhesive disc (adhesive film, agar-coated carrier, etc.), which is passed directly over the cut-out object for a few μm, can be used to catch the catapulted object. This adhesive flapper 20 is then thrown from the robot arm 22 in a suitable container (Fig. 3), and the robot arm 22 searches a new gluons (see Fig. 2).

Reference list

1
2 upright microscope
3 inverted microscope
4 slides
5
6 holder / device
7
8 microscope stage / stage
9
10 biol. Object (s)
11
12 lens
13 focused laser beam
14 trajectory
15
16 second holder
17th
18 collecting vessel
19th
20 adhesive flaps
21
22 robotic arm

Claims (2)

1. A method for separating and sorting biological objects on a planar carrier ( 4 ) on which the selected biological objects ( 10 ) are located together with further biological mass, characterized in that at least one self-contained strip-shaped part of the selected biological Object ( 10 ) surrounding further biological mass is removed by a laser beam ( 13 ), so that the selected biological object ( 10 ) is freed from its surroundings, and then the freed object ( 10 ) located on the carrier ( 4 ) by a another laser shot / laser-induced transport process is catapulted away from the carrier ( 4 ) to a collecting device ( 18 , 20 ) (trajectory 14 ). 2. Device for performing the method according to claim 1, characterized by

• - A microscope arrangement ( 2 ) for observing biological objects such as tissue sections on a slide ( 4 )
• a laser arrangement for generating a focused laser beam ( 13 ) for preparing one or more selected biological objects ( 10 ) on the slide ( 4 ), the laser arrangement and slide being arranged so as to be displaceable relative to one another,
• - And a collecting device ( 18 or 20 ) in the direction of the laser beam behind the slide ( 4 ) for collecting the selected objects ( 10 ), which after the preparation of the same by a laser-induced transport process (a "laser shot") from the slide ( 4 ) are catapulted away onto the catching device ( 18 or 20 ) (trajectory 14 ).