Method for the collision-free positioning of a micromanipulator tool

1. A method for the collision-free positioning of a micromanipulation tool relative to a sample carrier with the aid of a microscope, comprising the following steps:

a) determining the focal position Z p of at least one point P on the surface of the sample carrier by focusing a microscope objective with a first numerical aperture NA 1 on the at least one point;

b) positioning the micromanipulation tool on the optical axis of the microscope;

c) determining the focal position Z M of the micromanipulation tool by focusing the microscope objective or a second microscope objective with a second numerical aperture NA 2 smaller than the first numerical aperture NA 1 onto the tip of the micromanipulation tool;

d) calculating a lowering distance Z A (P) taking into account a predetermined tolerance profile ΔZ by the following equation:

Z A ( P )= Z M −Z P −ΔZ

where

Δ Z=ΔZ M +ΔZ P

wherein ΔZ M and ΔZ p is a predetermined tolerance in the determination of the position of the manipulation tool and the sample carrier, respectively;

e) positioning the micromanipulation tool at the point P by lowering by the lowering distance Z A (P).

2. The method according to claim 1 , wherein before carrying out step e), the steps c) and d) are repeated with a respective further, i-th microscope objective (i>1 and i>2) with a respectively increasing numerical aperture (NA i >NA i−1 ).

3. The method according to claim 1 , wherein the tolerance ΔZ M of the manipulation tool and/or the tolerance ΔZ p of the sample carrier is given by the depth of focus d tief,.i of the respective i-th objective used to determine its position, according to the following equation:

Δ

⁢

⁢

Z

M

,

P

=

d

tief

,

i

=

λ

·

n

NA

i

2

+

n

M

i

·

NA

i

·

e

i

=

1

,

2

,

…

where λ is the wavelength of the observation light, n is the refractive index of the medium between the microscope objective and sample carrier, M i is the magnification of the i-th objective, and e is the resolving power of a video camera used for the observation.

4. The method according to claim 1 , wherein before step e), the steps a) to d) are carried out for a plurality of predetermined points P j (j=1, 2, . . . ) on the surface of the sample carrier, the corresponding lowering distance Z A (P j ) for each point P j is stored in a database, and step e) is carried out for each point P j taking into account the stored lowering distance Z A (P j ).

5. The method according to claim 1 ,

wherein before step e), the steps a) to c) are carried out for three points P 1 , P 2 , P 3 on the surface of the sampler carrier that do not lie on a straight line, from whose determined focal positions Z p1 , Z p2 , Z p3 an equation of a plane E(P) is calculated, and in step d) the lowering distance Z A (P j ) for an arbitrary point P j on the surface of the sample carrier is determined by the equation

Z A ( P j )= Z M −E ( P j )−Δ Z.

6. The method according to claim 1 , wherein in step c) the position of the tip of the manipulation tool is determined as focal position Z M .

7. The method according to claim 6 , wherein in step c) a rotation angle and a tilt angle of the manipulation tool are compensated by carrying out the following steps:

c1) determining the focal position Z Q1 of a first point Q 1 on the tip of the manipulation tool by focusing on the point Q 1 ,

c2) rotating the manipulation tool about its axis and readjusting the microscope objective in order to focus the point Q 1 until the focal position Z Q1 of the point Q 1 and a focal position Z Q2 of a second point Q 2 on the tip of the manipulation tool are identical,

c3) determining the focal position Z Q3 of a third point Q 3 on the tip of the manipulation tool by focusing on the point Q 3 ,

c4) tilting the manipulation tool about an axis parallel to the plane of the sample carrier and readjusting the microscope objective in order to focus the point Q 3 until the focal positions Z Q1 , Z Q2 and Z Q3 are identical,

c5) fixing the focal position of the manipulation tool Z A =Z Q1 .

8. The method according to claim 1 , wherein in step a) in addition the focal position Z cap of a point R on the surface of a receiving vessel is determined by focusing the first microscope objective onto the point R.

9. The method according to claim 1 , wherein the point or points on the surface of the sample carrier and/or of the receiving vessel are approached by displacing it with the aid of a positioning table.

10. The method according to claim 1 ,

wherein the determination of the focal position is carried out with the aid of an optical distance measurement system.