DATA ACQUISITION METHOD USING A LASER SCANNER

• US 20110006219A1
• Filed: 02/13/2009
• Published: 01/13/2011
• Est. Priority Date: 02/21/2008
• Status: Active Grant

• Current Assignee: Tecan Trading AG (Tecan Group AG)
• Original Assignee: Tecan Trading AG (Tecan Group AG)
• Inventors: Fuchs, Wolfgang, Gfrörer, Andreas, Schausberger, Alois
• Granted Patent: US 8,222,615 B2
• Time in Patent Office: Days
• US Class Current: 250/459.1
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 21/278 Constitution of standards
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/104 Mechano-optical scan, i.e. object and beam moving
• CPC Class Codes: G01N 2201/11 Monitoring and controlling the scan
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/26 Stages; Adjusting means therefor

Abstract

The invention relates to a data acquisition method using a laser scanner for the pixel-precise imaging of fluorescent samples which are on object carriers and have been treated with fluorescent dyes. Such a laser scanner comprises a sample table; at least one laser and a first optical system for providing at least one laser beam for exciting the fluorescent samples; a scanner head (50) having an optical deflecting element for scanning this sample in at least one direction of movement (75); a first lens; a second optical system for forwarding emission beams, which are triggered by the laser beams on the sample and are deflected by the first lens and the deflecting element, to at least one detector; a position encoder (91) which emits position encoder signals (92) which indicate the instantaneous location of the scanner head (50) in relation to a zero point; an electronic element for filtering the detector signals (93) with a defined time constant; and an A/D converter for digitizing the filtered detector signals (93). The data acquisition method according to the invention is characterized in that the filtered detector signals (93) from the A/D converter and the position encoder signals (92) are acquired independently, in a parallel manner and continuously by a computer unit or a controller (40) and are related to a common time base (94), wherein the A/D conversion is carried out so often that each pixel (95) of an image is always assigned more than one data point of the A/D converter.

Claims

1. A data acquisition method using a laser scanner (1) for the pixel-precise imaging of samples, which are located on slides (8) and treated using fluorescent pigments, this laser scanner (1) comprising:

(a) a sample table (2) having a receptacle (34) for slides (8) in a sample plane (49);

(b) at least one laser (51, 52) and a first optical system (53) for providing at least one laser beam (54, 55) for exciting the fluorescent samples;

(c) a motor-driven scanner head (50), which moves nonlinearly in space, having a first optical deflection element (56) for deflecting the laser beams (54, 55) toward the sample and for scanning this sample in at least one movement direction (75);

(d) a first objective (57) for focusing the laser beams (54, 55) on the sample in the plane (49);

(e) a second optical system (58) for relaying emission beam bundles (59, 60), which are triggered by the laser beams (54, 55) on the sample and are deflected by the first objective (57) and the deflection element (56), to at least one detector (61);

(f) a position encoder (91), which emits position encoder signals (92), which indicate the instantaneous whereabouts of the scanner head (50) in relation to a zero point;

(g) an electronic element for filtering detector signals (93) of the detector (61) using a defined time constant; and

(h) an A/D converter for digitizing the filtered detector signals (93),

wherein the scanner head (50) is displaced on a linear guide (68), which defines the movement direction (75) of the scanner head (50),

wherein the filtered detector signals (93) of the A/D converter and the position encoder signals (92) are acquired chronologically in parallel and continuously but independently of one another by a computer unit and/or a controller (40) and related to a common timebase (94), the A/D conversion being performed with sufficient frequency that more than one data point of the A/D converter is always associated with each pixel (95) of an image,

and wherein the detector signal (93) generated by a detector (61) is digitized by the A/D converter of this controller (40), the moment of the corresponding position encoder signal (92) being associated with each conversion point of the A/D converter via the timebase (94), and each digitization being triggered in continuous progression by the timebase (94) and being performed by the controller (40) at a constant and equal time for all data acquisition intervals (Δ

d).

3. The data acquisition method according to claim 1,

wherein the signal filtering is executed using a passive RC element, the time constant selected for the RC element being in the order of magnitude of one data acquisition interval (Δ

d).

4. The data acquisition method according to claim 1,

wherein the signal filtering is executed using a passive RC element, the time constant selected for the RC element being a part of the order of magnitude of one pixel (95).

5. The data acquisition method according to claim 3,

wherein the intensity of the pixel (95) is calculated as the mean value of all data points which lie within time marks (96) for this pixel (95).

6. The data acquisition method according to claim 5,

wherein, in addition, the measured values of data acquisition intervals lying incompletely between the time marks of the pixel are proportionally interpolated.

7. The data acquisition method according to claim 1,

wherein a pixel resolution which is 2.0 μ

m or better is achieved using the laser scanner (1) when imaging fluorescent samples located on slides (8) and treated using fluorescent pigments.

8. The data acquisition method according to claim 7,

wherein pixels (95) having a size of 2 μ

m are imaged with at least two individual detector signals (93) using the laser scanner (1).

10. The data acquisition method according to claim 1,

wherein the connecting rod attachment point (69) of the scanner head (50) is situated in the movement direction (75) on a line with a mass center of gravity (74) of the scanner head (50).

11. The data acquisition method according to claim 1,

wherein the scanner head (50) defines a scanning plane (76) with its optical deflection element (56) and its movement direction (75), the scanner head (50) comprising a scale (77), which is situated at a distance to a linear measuring system (78) of the laser scanner (1) and in this scanning plane (76).

12. The data acquisition method according to claim 1,

wherein the scanner head (50) comprises a scale (77), which is situated at least in proximity to a main plane (107) of the first objective (57).

13. The data acquisition method according to claim 1,

wherein the scanner apparatus (72) comprises a counter oscillator (73), which is fastened so it is displaceable on a linear guide and has a connecting rod attachment point (69′

), which is connected via a connecting rod (70′

) to the drive (71) of the scanner apparatus (72).

14. The data acquisition method according to claim 1,

wherein the sample plane (49) is situated essentially horizontally.

15. The data acquisition method according to claim 1,

wherein the movement direction (75) of the scanner head (50) defines an X axis or scanning axis of a Cartesian coordinate system and the sample table (2) is moved linearly in a Y direction situated perpendicularly thereto.

16. The data acquisition method according to claim 1,

wherein two laser beams (54, 55) of different wavelengths, which are oriented parallel to one another and run parallel to the sample plane (49), are provided using the at least one laser (51, 52) and the first optical system (53) and deflected toward the sample using an optical deflection element (56) of a scanner apparatus (72).

17. The data acquisition method according to claim 16,

wherein these laser beams (54, 55) are focused on the sample in the plane (49) using the first objective (57), after which the emission beam bundles (59, 60) of different wavelengths, which are triggered by the laser beams (54, 55) and are deflected by the first objective (57) and the deflection element (56) in a direction essentially parallel to the plane (49), are conducted using the second optical system (58) to two detectors (61, 61′

) and acquired by these detectors (61, 61′

).

18. The data acquisition method according to claim 17,

wherein a wedge-shaped dichroic mirror (62) having front and rear dichroic surfaces (63, 64), which are situated at an intermediate angle (â

) to one another, is used as part of the optical deflection element (56), the wedge-shaped dichroic mirror (62) being set so that the two laser beams (54, 55) are each reflected on one of the surfaces (63, 64), and the wedge-shaped dichroic mirror (62) causing a spatial separation of the two resulting focal points (65) and of the two emission beam bundles (59, 60) deflected in the direction of the detectors (61, 61′

) by the intermediate angle (β

).

19. The data acquisition method according to claim 17,

wherein an optical deflection element (56) implemented as a pentamirror configuration (66) having a wedge-shaped dichroic mirror (62) and a single mirror (67) is used, this pentamirror configuration (66) correcting tilts of the scanner head (50) around a Y axis running perpendicular to the scanning axis (75) so that the resulting focal points (65) do not change their current position in the sample plane (49).

20. The data acquisition method according to claim 18,

wherein the deflection of the scanner head (50) in the X axis (75) is measured using a scale (77), which is situated at a distance to a linear measuring system (78) of the laser scanner (1) in this scanning plane (76).

21. The data acquisition method according to claim 18

wherein the deflection of the scanner head (50) in the X axis (75) is measured using a scale (77), which is situated at least in proximity to the main plane (107) of the first objective (57).

2. (canceled)

9. (canceled)