Laser microdissection method and apparatus

• US 9,279,749 B2
• Filed: 05/05/2014
• Issued: 03/08/2016
• Est. Priority Date: 09/09/2004
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Donovan, Brian, Baer, Thomas
• Primary Examiner(s): Claytor, Renee
• Assistant Examiner(s): FERNANDEZ, SUSAN EMILY
• Application Number:
  US14/270,275
• Publication Number:
  US 20140242630A1
• Time in Patent Office: 673 Days
• US Class Current: 1/1
• CPC Class Codes: G01N 1/28 Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Qmounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 ; laboratory gas handling apparatus B01L5/00
• 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 1/286 involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising microtomes G01N1/06; pulverising in general B02C; mixing in general B01F
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 1/44 Sample treatment involving radiation, e.g. heat
• CPC Class Codes: G01N 2001/2826 Collecting by adsorption or absorption
• CPC Class Codes: G01N 2001/2833 Collecting samples on a sticky, tacky, adhesive surface
• CPC Class Codes: G01N 2001/284 using local activation of adhesive, i.e. Laser Capture Microdissection
• CPC Class Codes: G01N 2001/2886 Laser cutting, e.g. tissue catapult
• 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. In one variation, targeted biological material is manually or automatically selected and a transfer film is placed in juxtaposition to the location of an interior of a cut path. In another variation, a sample of biological material is mounted onto a polymer membrane which is then placed onto a substrate. Targeted biological material is manually or automatically selected and a transfer film is placed in juxtaposition with the targeted biological material on the side of the biological material. In yet another variation, a sample of biological material is mounted onto a polymer membrane which is then inverted onto a substrate. Targeted biological material is manually or automatically selected and a transfer film is placed in juxtaposition with the targeted biological material on the side of the polymer membrane. Then, an UV laser cuts along a cut path around the targeted portions of biological material in a closed cut path or a substantially closed cut path defining an interior and an exterior portion of the tissue sample. In a substantially closed cut path, bridges are left spanning the interior of the cut path and the exterior of the cut path. An IR laser activates at least a portion of the transfer film such that the transfer film in the vicinity of targeted portion adheres to the biological material interior to the cut path. The transfer film is then removed separating the targeted portions of biological material which are adhered to the transfer film from the remaining portion of the tissue sample.

Claims

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

providing a first substrate having an upper surface;

providing a membrane having an upper surface and a lower surface;

applying a layer of a biological material to upper surface of the membrane;

placing the membrane in contact with the first substrate such that the lower surface of the membrane contacts the upper surface of the first substrate;

providing a second substrate having a surface with a transfer film disposed thereon, wherein the transfer film has adhesive characteristics upon activation by electromagnetic energy;

identifying at least one targeted portion of the biological material to be microdissected;

bringing the transfer 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 the biological material;

activating a first laser source so as to describe at least one closed or substantially closed path around the at least one targeted portion of the biological material to be microdissected, the first laser source eroding the biological material along the described path and defining an interior portion of the biological material and an exterior portion of the biological material;

activating a second laser source and directing the second laser source at the interior portion of the biological material so as to activate at least one region of the transfer film so that the at least one activated region of the transfer film adheres to the interior portion of the biological material; and

separating the transfer film and the at least one adhered targeted portion of the biological material from the first substrate.

2. The method of claim 1 wherein the first laser source is a UV laser source and the second laser source is an IR laser source.

3. 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 portion to the exterior portion wherein the interior portion is joined to the surrounding exterior biological material at the at least one bridge.

4. The method of claim 3 wherein the step of separating the second substrate with its attached transfer film and the at least one adhered targeted portion of the biological material from the remaining layer of the biological material includes the step of breaking the at least one bridge.

5. The method of claim 3 wherein the step of activating the second laser source includes the step of activating the second laser source in between bridges.

6. The method of claim 1 wherein the step of activating the second laser source includes the step of activating the second laser source so as to describe at least one closed or partially closed curve at the interior portion.

7. The method of claim 1 wherein the step of activating the second laser source includes the step of activating the second laser source more than one time in the interior portion.

8. The method of claim 1 wherein the step of bringing the transfer 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 the biological material includes spacing a substantial portion of the transfer film away from the biological material by a distance sufficient for promoting adhesion of the transfer film to the biological material upon activation of the transfer film by the second laser source.

9. The method of claim 1:

wherein the at least one targeted portion includes desired and undesired biological material; and

wherein the at least one activated region of transfer film adheres to at least one portion of the undesired biological material.

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

providing a first substrate having an upper surface;

providing a membrane having an upper surface and a lower surface;

applying a layer of a biological material to upper surface of the membrane;

placing the membrane in contact with the first substrate such that the biological material contacts the upper surface of the first substrate and the upper surface of the membrane faces the upper surface of the first substrate;

providing a second substrate having a surface with a transfer film disposed thereon, wherein the transfer film has adhesive characteristics upon activation by electromagnetic energy;

applying the transfer film to the surface of the second substrate;

identifying at least one targeted portion of the biological material to be microdissected;

placing the transfer film into juxtaposition with the first substrate on the side of the membrane in the location of the at least one targeted portion of the biological material;

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

the first laser source eroding the biological material and membrane along the described path and defining an interior portion of the biological material and an exterior portion of the biological material;

activating a second laser source and directing the second laser source at the interior portion of the biological material so as to activate at least one region of the transfer film so that the at least one activated region of the transfer film adheres to the interior portion of the biological material; and

separating the second substrate with its attached transfer film, the membrane, and the at least one adhered targeted portion of the biological material from the first substrate.

11. The method of claim 10 wherein the first laser source is a UV laser source and the second laser source is an IR laser source.

12. The method of claim 10 wherein activating the first laser source includes describing a substantially closed path such that there remains at least one bridge between the interior of the biological material to the exterior of the biological material, wherein the interior of the biological material is joined to the surrounding exterior of the biological material at the at least one bridge.

13. The method of claim 10 wherein the step of bringing the transfer film into juxtaposition with the first substrate on the side of the membrane in the location of the at least one targeted portion of the biological material includes spacing a substantial portion of the transfer film away from the membrane by a distance sufficient for promoting adhesion of the transfer film to the membrane upon activation of the transfer film by the second laser source.

14. The method of claim 10 wherein the step of 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 transfer film so that the at least one activated region of the transfer film adheres to the interior portion of the biological material includes:

keeping the second substrate in a fixed position relative to the membrane; and

bringing the transfer film into contact with the membrane for adhesion to the membrane.

15. The method of claim 10:

wherein the at least one targeted portion includes desired and undesired biological material; and

wherein the at least one activated region of transfer film adheres to at least one portion of the undesired biological material.

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

providing a first substrate having an upper surface;

providing a membrane having an upper surface and a lower surface;

applying a layer of a biological material to upper surface of the membrane;

placing the membrane in contact with the first substrate such that the lower surface of the membrane contacts the upper surface of the first substrate;

providing a second substrate having a surface with a transfer film disposed thereon, wherein the transfer film has adhesive characteristics upon activation by electromagnetic energy;

identifying at least one targeted portion of the biological material to be microdissected;

bringing the transfer 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 the biological material;

activating a laser source and directing the laser source at the transfer film so that at least one activated region of the transfer film adheres to the biological material; and

separating the second substrate with its attached transfer film and the at least one adhered targeted portion of the biological material from the first substrate;

wherein the at least one targeted portion includes desired and undesired biological material;

wherein the at least one activated region of transfer film adheres to at least one portion of the undesired biological material.

17. The method of claim 16 wherein the laser source is an IR laser source.