Laser scanning microscope for scanning along a 3D trajectory

• US 9,081,173 B2
• Filed: 07/14/2009
• Issued: 07/14/2015
• Est. Priority Date: 07/15/2008
• Status: Active Grant

• Current Assignee: Femtonics Kft.
• Original Assignee: Femtonics Kft.
• Inventors: Vizi, E. Szilveszter, Katona, Gergely, Rozsa, J. Balazs
• Primary Examiner(s): NGUYEN, THONG Q
• Application Number:
  US12/737,426
• Publication Number:
  US 20110211254A1
• Time in Patent Office: 2,191 Days
• Field of Search: 359/368, 359/383, 359/385, 359391-393, 3591961-1991
• US Class Current: 1/1
• 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

A laser scanning microscope is provided with a laser beam focusing device, a drive for the focusing device, a laser beam deflector, and a control system that coordinates movement of the focusing device with that of the laser beam deflector.

Claims

1. Laser scanning microscope (10) comprising

focusing means (15) having a focal plane (29) and an optical axis 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 a position of the focal plane (29), and

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

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

providing a periodical drive signal having a frequency between 50 and 200 Hz for the drive means (18),

displacing the at least one optical element of the focusing means (15) along its optical axis with the drive means (18) in response to the periodical drive signal, 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, and

calculating an XY drive signal for the deflecting means (14) using the response function (z(t)) which takes into account a shape distortion of the response function (z(t)) with respect to the periodical drive signal,

generating the XY drive signal for the deflecting means (14) in order to move a 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-electric device (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 control system is configure to provide a sinusoidal voltage signal for the drive means (18).

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

) or accousto-optical deflectors.

9. Method for scanning a sample (22) along a 3D trajectory (48) at a frequency between 50 to 200 Hz using a laser scanning microscope (10) having

focusing means (15) having a focal plane (29) and an optical axis 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 a position of the focal plane (29), and

deflecting means (14) for deflecting the laser beam (13), which comprises the steps of:

providing a periodical drive signal having a frequency between 50 and 200 Hz for the drive means (18),

displacing the at least one optical element of the focusing means (15) along its optical axis with the drive means (18) in response to the periodical drive signal,

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, and

calculating an XY drive signal for the deflecting means (14) using the response function (z(t)) which takes into account a shape distortion of the response function (z(t)) with respect to the periodical drive signal,

generating the XY drive signal for the deflecting means (14) in order to move a focal volume (30) of the laser beam (13) along the 3D trajectory (48) within the sample (22).

10. The method according to claim 9, wherein a confocal laser scanning microscope (10) or a two-photon laser scanning microscope (10) is used.

11. The method according to claim 9, wherein the focusing means (15) comprises an objective (16) as optical element and the drive means (18) comprises a piezo-electric device (18′

) for displacing the objective (16).

12. The method according to claim 9, wherein the drive signal provided for the drive means (18) is a sinusoidal voltage signal.

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

14. The laser scanning microscope according to claim 13, 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.

15. The method according to claim 9, wherein the deflecting means (14) comprises galvanometric scanning mirrors (14′

) or accousto-optical deflectors.

16. The method according to claim 9, 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).