Method for high spatial resolution examination of samples

Abstract

A method for high spatial resolution examination of samples, preferably by using a laser scanning fluorescence microscope, the sample (1) to be examined comprising a substance that can be repeatedly converted from a first state (Z1, A) into a second state (Z2, B), the first and the second states (Z1, A; Z2, B) differing from one another in at least one optical property, comprising the following steps: a) the substance is brought into the first state (Z1, A) by means of a switching signal (2) in a sample region (P) to be recorded,b) the second state (Z2, B) is induced by means of an optical signal (4), spatially delimited subregions being specifically excluded within the sample region (P) to be recorded,c) the remaining first states (Z1, A1, A2, A3) are read out by means of a test signal (7), andd) steps a) to c) are repeated, the optical signal (4) being displaced upon each repetition in order to scan the sample (1), is defined in that the individual steps a) to d) are carried out in a sequence adapted to the respective measuring situation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred refinements and developments of the teaching are also explained in general in conjunction with explanations of the preferred exemplary embodiment and with the aid of the drawing, in which the sole

figure shows a schematic illustration of an exemplary embodiment of a method for high spatial resolution examination of samples.

Claims

1. A method for high spatial resolution examination of samples, preferably by using a laser scanning fluorescence microscope, the sample (1) to be examined comprising a substance that can be repeatedly converted from a first state (Z1, A) into a second state (Z2, B), the first and the second states (Z1, A; Z2; B) differing from one another in at least one optical property, comprising the following steps: a) the substance is brought into the first state (Z1, A) by means of a switching signal (2) in a sample region (P) to be recorded,b) the second state (Z2, B) is induced by means of an optical signal (4), spatially delimited subregions being specifically excluded within the sample region (P) to be recorded,c) the remaining first states (Z1, A1, A2, A3) are read out by means of a test signal (7), andd) steps a) to c) are repeated, the optical signal (4) being displaced upon each repetition in order to scan the sample (1),wherein the individual steps a) to d) are carried out in a sequence adapted to the respective measuring situation.

2. The method as claimed in claim 1, wherein a subcycle comprising a subset of steps a) to d) is repeatedly executed within the overall cycle comprising steps a) to d).

3. The method as claimed in claim 2, wherein the repeatedly executed subcycle comprises steps b) and c).

4. The method as claimed in claim 2, wherein the repeatedly executed subcycle comprises steps a), b) and c).

5. The method as claimed in claim 1, wherein measuring signals (8) resulting from the reading out of the first states (Z1, A1, A2, A3) are respectively detected only during and/or shortly after the emission of a test signal (7).

6. The method as claimed in claim 1, wherein a detector for detecting measuring signals (8) is synchronized with the respective sequence of the steps.

7. The method as claimed in claim 1, wherein a shutter arranged upstream of a detector for detecting measuring signals (8) is synchronized with the respective sequence of the steps.

8. The method as claimed in claim 6, wherein a CCD or EMCCD camera is used as detector.

9. The method as claimed in claim 6, wherein a photomultiplier or an avalanche photodiode (APD) is used as detector.

10. The method as claimed in claim 6, wherein a detector array, preferably an APD array, is used as detector.

11. The method as claimed in claim 1, wherein steps a) and b) are executed in a fashion that is simultaneous or at least partially temporally overlapping.

12. The method as claimed in claim 1, wherein the imaging is advanced by means of galvanometer scanners, acoustooptic deflectors, MEMS or piezomechanical elements.

13. The method as claimed in claim 12, wherein the respective sequence of the steps is synchronized with the pixel clock and/or the advancements of the imaging.

14. The method as claimed in claim 13, wherein the synchronization is implemented by means of AOTFs and/or AOMs and/or electronically and/or by means of mechanical shutters.

15. The method as claimed in claim 1, wherein a prescribable delay is set between the individual steps.

16. The method as claimed in claim 15, wherein the delays between the individual steps, and/or the duration of the individual steps are themselves set to be of different length.