Method for autofocusing a microscope at a correct autofocus position in a sample

1. A method for autofocusing a microscope at a correct autofocus position in a sample comprising the steps:

generating a reference pattern by an autofocus light device,

projecting, by the autofocus light device, the reference pattern through a microscope objective and towards a sample, whereby the reference pattern is backscattered by at least two interfaces being located at or close to the sample,

projecting the backscattered reference pattern through the microscope objective and towards a detector which provides spatial resolution,

obtaining, by the detector, a superposition of a number of detection patterns, each detection pattern related to one of the at least two interfaces,

analyzing the superposition of detection patterns to identify at least one autofocus detection pattern related to at least one of the interfaces, and

analyzing the at least one autofocus detection pattern to determine the direction and/or magnitude of deviation of the microscope objective's current focus position from a correct focus position.

2. The method according to claim 1 , wherein obtaining a superposition of detection patterns further comprises obtaining a superposition of detection patterns wherein a position, a size a shape, or an X:Y aspect ratio of each detection pattern on the detector depends on a position of the at least one interface relative to the microscope objective.

3. The method according to claim 1 , further comprising analyzing a distribution of autofocus light intensity to further define the autofocus detection pattern.

4. The method according to claim 1 , wherein analyzing the superposition of detection patterns to identify at least one autofocus detection pattern further comprises using a priori knowledge of a geometry of the sample and/or a geometry close to the sample.

5. The method according to claim 1 , wherein analyzing the superposition of detection patterns to identify at least one autofocus detection pattern further comprises using a priori knowledge of the number of interfaces and of their spatial order.

6. The method according to claim 1 , wherein generating a reference pattern further comprises generating a reference pattern using an autofocus light device comprising an astigmatic lens.

7. The method according to claim 1 , wherein generating a reference pattern further comprises generating a reference pattern comprising a beam path that is tilted relative to an optical axis of the microscope objective.

8. The method according to claim 1 , wherein analyzing the at least one autofocus detection pattern further comprises:

comparing a size, shape or position of the autofocus detection pattern to a size, shape or position of an autofocus detection pattern of a correct autofocus position; and

using the comparison to determine the direction and/or magnitude of deviation of the microscope objective's current focus position from the correct focus position.

9. A system for autofocusing a microscope at a correct autofocus position in a sample, the system comprising:

a microscope objective for observing a sample;

an autofocus light device for projecting a reference pattern through the microscope objective towards the sample, wherein the reference pattern is backscattered by at least two interfaces located at or close to the sample;

a detector obtaining the backscattered reference pattern as a superposition of a number of detection patterns, wherein each detection pattern relates to one of the at least two interfaces; and

a computer executing steps for:

identifying at least one autofocus detection pattern related to at least one of the at least two interfaces by analyzing the superposition of the detection patterns, and

determining a direction or magnitude of deviation of the microscope objective's current focus position from a correct focus position by analyzing the autofocus detection pattern.

10. The system of claim 9 , wherein the computer further executes the step of focusing a lens of the microscope objective based on the determined direction and magnitude of deviation.

11. The system of claim 9 , wherein the autofocus light device comprises an astigmatic lens.

12. The system of claim 9 , wherein the autofocus light device projects a reference pattern with a beam path that is tilted relative to an optical axis of the microscope objective.

13. The system of claim 9 , wherein the one of the at least two interfaces comprises an interface close to the microscope objective.

14. The system of claim 13 , wherein the autofocus detection pattern comprises the smallest detection pattern obtained by the detector.

15. The system of claim 9 , wherein the one of the at least two interfaces comprises an interface between a transparent substrate supporting the sample and air between the transparent substrate and the microscope objective.

16. The system of claim 9 , wherein the one of the at least two interfaces comprises an interface between a transparent substrate supporting the sample and the sample.

17. The system of claim 9 , wherein the at least two interfaces comprises three interfaces, and wherein the detection pattern relates to a middle interface of the three interfaces.