Automated microdissection instrument

• US 20060139621A1
• Filed: 09/26/2005
• Published: 06/29/2006
• Est. Priority Date: 11/05/2001
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Smith, Michael A., Wessel, Alan E., Dawson, George M., Barker, Craig S., Donovan, Brian W., Baer, Thomas M., Youngquist, Michael G., Leclerc, Norbert H.
• Granted Patent: US 8,722,357 B2
• Time in Patent Office: Days
• US Class Current: 356/36
• CPC Class Codes: G01B 11/002 for measuring two or more coordinates
• CPC Class Codes: G01N 1/2813 Producing thin layers of samples on a substrate, e.g. smearing, spinning-on G01N1/30 takes precedence
• CPC Class Codes: G01N 2001/282 with mapping; Identification of areas; Spatial correlated pattern
• CPC Class Codes: G01N 2001/284 using local activation of adhesive, i.e. Laser Capture Microdissection
• CPC Class Codes: Y10T 156/10 Methods of surface bonding and/or assembly therefor
• CPC Class Codes: Y10T 156/1054 and simultaneously bonding [e.g., cut-seaming]

Abstract

Systems and methods for automated laser microdissection are disclosed including automatic slide detection, position detection of cutting and capture lasers, focus optimization for cutting and capture lasers, energy and duration optimization for cutting and capture lasers, inspection and second phase capture and/or ablation in a quality control station and tracking information for linking substrate carrier or output microdissected regions with input sample or slide.

Claims

1. A method for laser microdissection comprising the steps of:

providing a first substrate having a surface;

applying a layer of biological material to the surface of the first substrate;

providing a polymer layer;

identifying at least one targeted portion of biological material located on the first substrate;

bringing the polymer layer into juxtaposition with the first substrate on the side of the biological material in the location of the at least one targeted portion of biological material;

providing at least one laser source;

activating the at least one laser source so as to describe at least one closed or substantially closed path around the at least one targeted portion of biological material or directly at the least one targeted portion of biological material;

transferring at least one portion of biological material from the layer of biological material to the polymer layer;

moving the polymer layer to a quality control station;

identifying at least one portion of biological material that is present on the polymer layer while the polymer layer is located in the quality control station;

activating the at least one laser source and directing it at the at least one portion of identified biological material that is present on the polymer layer while the polymer layer is located in the quality control station.

2. The method of claim 1 wherein the step of activating the at least one laser source includes the step of ablating undesirable tissue.

3. The method of claim 1 further including the step of providing a second laser source;

and

wherein the step of transferring at least one portion of biological material from the layer of biological material to the polymer layer includes activating a second laser and directing the second laser at a portion of the biological material.

4. The method of claim 3 wherein the second laser source is an IR laser source.

5. The method of claim 3 wherein the second laser source is a UV laser source.

6. The method of claim 1 further including the steps of:

providing a second substrate having a surface; and

applying the polymer film to the surface of the second substrate.

7. The method of claim 6 further including the step separating the second substrate with its attached polymer film and the at least one adhered targeted portion of biological material from the remaining layer of biological material.

8. The method of claim 6 wherein the polymer film is a transfer film having adhesive characteristics upon activation by electromagnetic energy.

9. The method of claim 1 wherein the first substrate is a polyethylene naphalate (PEN) membrane or a polyethylene terephthalate (PET) membrane.

10. The method of claim 1 further including the steps of:

providing a second laser source; and

activating the second laser source and directing the second laser source at the interior of the at least one described path so as to activate at least one region of the polymer film so that the at least one activated region of polymer film adheres to the at least one interior portion of biological material.

11. The method of claim 1 wherein the first laser source cuts the biological material along the described path and defines an interior and exterior.

12. The method of claim 1 wherein the first laser source is a UV laser source.

13. The method of claim 1 wherein the second laser source is an IR laser source.

14. The method of claim 1 wherein the step of activating the first laser source includes describing a substantially closed path such that there remains at least one bridge between the interior to the exterior wherein the interior is joined to the surrounding exterior biological material at the at least one bridge.

15. The method of claim 14 wherein the step of transferring at least one portion of biological material from the layer of biological material to the polymer layer includes the step of breaking the at least one bridge.

16. The method of claim 1 wherein the step of bringing the second substrate with the polymer film into juxtaposition with the first substrate on the side of the biological material in the location of the at least one targeted portion of biological material includes contacting the polymer film to the biological material.

17. The method of claim 1 wherein the step of bringing the second substrate with the polymer film into juxtaposition with the first substrate on the side of the biological material in the location of the at least one targeted portion of biological material includes spacing a portion of the polymer film away from the biological material by a distance sufficient for promoting adhesion of the polymer film to the biological material upon activation of the polymer film by the at least one laser source.

18. A method for automatically determining the location of a laser beam projection on a worksurface area of a laser microdissection instrument that is operatively coupled to a microprocessing device and a digital image acquisition system containing a digital image sensor, the method comprising the steps of:

emitting the laser beam at an intensity at a level sufficient to be detected by the digital image sensor;

detecting the light intensity of the laser beam by the digital image sensor;

determining the pixel location of the increased light intensity on the digital image sensor;

converting the pixel location of the increased light intensity on the digital image sensor to a coordinate location corresponding to the worksurface area; and

assigning the coordinate location to the location of the laser beam.

19. The method of claim 18 further including the step of turning off background illumination.

20. The method of claim 18 further including the step of moving the laser beam from the assigned coordinate location to a target location.

21. The method of claim 18 further including the step of increasing the intensity of the laser beam.

22. A method for optimizing the focus of a laser beam in a laser microdissection instrument comprising the steps of:

providing a laser microdissection instrument having a worksurface;

providing a first laser source and laser focusing lens on a first side of the worksurface;

providing an objective lens and image acquisition system on a second side of the worksurface;

providing a sample on the worksurface;

focusing the objective lens on the sample for a clear image of the sample acquired by the image acquisition system;

activating the first laser source to emit a laser beam directed at the sample;

focusing the laser beam from the first laser source by moving the laser focusing lens;

re-focusing the objective lens at least once on the sample by moving the objective lens a distance;

keeping the first laser source at the desired focus by moving the focusing lens by substantially the same distance that the objective lens was moved when re-focused.

23. The method of claim 22 wherein if the objective lens is moved toward the worksurface on re-focus, the step of keeping the first laser source at the desired focus includes moving the laser focusing lens away from the worksurface and if the objective lens is moved away from the worksurface on re-focus, the step of automatically keeping the first laser source at the desired focus includes moving the laser focusing lens toward the worksurface.

24. The method of claim 22 further including the step of changing the objective lens to a different objective lens.

25. The method of claim 24 further including the step of focusing the different objective lens after the step of changing the objective lens and keeping the focusing lens locked.

26. The method of claim 25 further including the steps of:

automatically re-focusing the different objective lens on the sample by moving the objective lens a distance;

automatically keeping the first laser source at the desired focus by moving the focusing lens by the same distance that the different objective lens was moved when re-focused.

27. The method of claim 22 wherein the first laser source is an IR laser.

28. A method for laser microdissection instrument comprising the steps of:

providing a first substrate having a transfer film attached;

providing a second substrate having biological material;

introducing the second substrate with the biological material into the laser microdissection instrument;

identifying at least one targeted portion of biological material on the second substrate;

bringing the first substrate into juxtaposition with the second substrate on the side of the biological material in the location of the at least one targeted portion of biological material;

providing a first laser source;

activating the first laser source to activate at least one region of the transfer film to adhere at least one portion of biological material to the transfer film;

transferring at least one portion of biological material from the second substrate to the transfer film and first substrate; and

recording at least one tracking information associated with the first substrate.

29. The method of claim 28 further outputting the at least one tracking information upon user request.

30. The method of claim 28 wherein the at least one tracking information includes information related to the historical location of the cap.

31. The method of claim 28 wherein the at least one tracking information includes information related the biological material.

32. The method of claim 28 wherein the step of recording at least one tracking information includes recording at least one tracking information associated with the first substrate linking the first substrate, the biological material, and the at least one second substrate from which the biological material was removed.

33. A digital microscope for observing a sample, comprising:

a worksurface for receiving a sample;

the worksurface intersecting a primary optical axis of the microscope;

a substrate receiving location on the worksurface for receiving a sample-bearing substrate;

the worksurface having a first opening in the substrate receiving location for alignment with the primary optical axis to permit pathing of light through the first opening in the worksurface;

a digital image acquisition system including an image sensor configured to automatically detect the presence of the substrate in the substrate-receiving location.

34. The digital microscope of claim 33 further including a quality control station.

35. The digital microscope of claim 34 wherein the quality control station is located on the worksurface;

the worksurface having a second opening in the location of the quality control station permitting the pathing of light through the second opening in the worksurface.

36. The digital microscope of claim 33 further including at least one laser source optically coupled to the microscope to path laser light along the primary optical axis for laser microdissection of the sample introduced on the sample-bearing substrate.

37. The digital microscope of claim 33 wherein the digital image acquisition system is configured to automatically detect the presence of the substrate in the substrate-receiving location by detecting a light intensity differential when a sample-bearing substrate is placed in the substrate receiving location.

38. The digital microscope of claim 33 wherein the digital image acquisition system is configured to capture an image of the sample after the presence of a sample-bearing surface is detected in the substrate-receiving location.