LASER SCANNING MICROSCOPE FOR SCANNING ALONG A 3D TRAJECTORY

• US 20110211254A1
• Filed: 07/14/2009
• Published: 09/01/2011
• Est. Priority Date: 07/15/2008
• Status: Active Grant

• Current Assignee: Femtonics Kft.
• Original Assignee: Femtonics Kft.
• Inventors: Katona, Gergely, Vizi, E. Szilveszter, Rozsa, J. Balazs
• Granted Patent: US 9,081,173 B2
• Time in Patent Office: Days
• US Class Current: 359/385
• CPC Class Codes: G02B 21/0048 scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• CPC Class Codes: G02B 21/006 focusing arrangements; selection of the plane to be imaged
• CPC Class Codes: G02B 21/0084 time-scale detection, e.g. strobed, ultra-fast, heterodyne detection

Abstract

The invention relates to a laser scanning microscope (10) having: focusing means (15) having a focal plane (29) and comprising at least one optical element for focusing a laser beam (13); drive means (18) for displacing the at least one optical element of the focusing means (15) for changing the position of the focal plane (29), and deflecting means (14) for deflecting the laser beam (13). The microscope comprises a control system (32) configured to carry out the steps of: providing a periodical drive signal for the drive means (18); obtaining time dependant displacement data of the at least one optical element of the focusing means (15) in response to the periodical drive signal of the drive means (18); providing a response function (z(t)) using the time dependant displacement data, calculating a drive signal for the deflecting means (14) using the response function (z(t)) to move the focal volume (30) of the laser beam (13) along a given 3D trajectory (48) within a sample to be examined. The invention further relates to a method for carrying out such a scanning operation along a 3D trajectory (48).

Claims

1. Laser scanning microscope (10) having

focusing means (15) having a focal plane (29) and comprising at least one optical element for focusing a laser beam (13),

drive means (18) for displacing the at least one optical element of the focusing means (15) for changing the position of the focal plane (29), and

deflecting means (14) for deflecting the laser beam (13),

characterised by a control system (32) configured to carry out the steps of

providing a periodical drive signal for the drive means (18),

obtaining time dependant displacement data of the at least one optical element of the focusing means (15) in response to the periodical drive signal of the drive means (18),

providing a response function (z(t)) using the time dependant displacement data,

calculating a drive signal for the deflecting means (14) using the response function (z(t)) to move the focal volume (30) of the laser beam (13) along a given 3D trajectory (48) within a sample to be examined.

2. The laser scanning microscope according to claim 1, wherein the microscope (10) is a two-photon laser scanning microscope.

3. The laser scanning microscope according to claim 1, wherein the microscope (10) is a confocal laser scanning microscope.

4. The laser scanning microscope according to claim 1, wherein the focusing means (15) comprises an objective (16) as optical element and the drive means (18) comprises a piezo positioner (18′

) configured to displace the objective (16).

5. The laser scanning microscope according to claim 1, wherein the focusing means (16) comprises a plurality of optical elements of which at least two is provided with drive means (18), the control system (32) providing periodical drive signals to each such drive means (18).

6. The laser scanning microscope according to claim 5, wherein the drive signals of the at least two optical elements are phase shifted relative to each other.

7. The laser scanning microscope according to claim 1, wherein the deflecting means (14) comprises galvanometric scanning mirrors (14′

) or accousto-optical deflectors.

8. The laser scanning microscope according to claim 1, wherein the control system is configure to provide a sinusoidal voltage signal for the drive means (18).

9. The laser scanning microscope according to claim 1 further comprising securing means for fixing the position of the sample (22) to be examined.

10. The laser scanning microscope according to claim 1, wherein the sample (22) is a biological specimen (22′

) and the securing means is a support grid (60) having mesh spaces (62) through which the biological specimen (22′

) can be nourished with a physiological solution.

11. Method for scanning a sample (22) along a 3D trajectory (48) using a laser scanning microscope (10) having

focusing means (15) having a focal plane (29) and comprising at least one optical element for focusing a laser beam (13),

drive means (18) for displacing the at least one optical element of the focusing means (15) for changing the position of the focal plane (29), and

deflecting means (14) for deflecting the laser beam (13),

characterised by the steps of:

providing a periodical drive signal for the drive means (18),

obtaining time dependant displacement data of the at least one optical element of the focusing means (15) in response to the periodical drive signal of the drive means (18),

providing a response function (z(t)) using the time dependant displacement data,

calculating a drive signal for the deflecting means (14) using the response function (z(t)) to move the focal volume (30) of the laser beam (13) along the 3D trajectory (48) within the sample (22).

12. The method according to claim 11, wherein a confocal laser scanning microscope (10) or a two-photon laser scanning microscope (10) is used.

13. The method according to claim 11, wherein the focusing means (15) comprises an objective (16) as optical element and the drive means (18) comprises a piezo positioner (18′

) for displacing the objective (16).

14. The method according to claim 11, wherein the deflecting means (14) comprises galvanometric scanning mirrors (14′

) or accousto-optical deflectors.

15. The method according to claim 11, wherein the drive signal provided for the objective drive means (18) is a sinusoidal voltage signal.

16. The method according to claim 11, further comprising the step of placing a living biological specimen as sample (22) on a microscope stage (20) comprising a support grid (60) having mesh spaces (62) and nourishing the specimen (22) with a physiological solution through the mesh spaces (62).