TRANSMISSION ELECTRON MICROSCOPY FOR IMAGING LIVE CELLS

• US 20120120226A1
• Filed: 11/17/2011
• Published: 05/17/2012
• Est. Priority Date: 11/17/2010
• Status: Active Grant

• Current Assignee: Anselm D.E. Jonge, Frederik D.E. Jonge, Niels D.E. Jonge, Vilija D.E. Jonge
• Original Assignee: Vanderbilt University
• Inventors: de Jonge, Niels
• Granted Patent: US 9,207,196 B2
• Time in Patent Office: Days
• US Class Current: 348/80
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: G01N 23/2204 Specimen supports therefor; Sample conveying means therefore
• CPC Class Codes: G01N 23/2251 using incident electron beams, e.g. scanning electron microscopy [SEM]
• CPC Class Codes: H01J 2237/2003 Environmental cells
• CPC Class Codes: H01J 2237/2004 Biological samples
• CPC Class Codes: H01J 2237/2802 Transmission microscopes
• CPC Class Codes: H01J 37/20 Means for supporting or positioning the objects or the material; Means for adjusting diaphragms or lenses associated with the support introducing the objects H01J37/18

Abstract

In one aspect, the present invention relates to a microfluidic chamber. In one embodiment, the microfluidic chamber has a first sub-chamber and at least one second sub-chamber. The first sub-chamber has a first window and a second window. Both the first window and the second window are transparent to electrons of certain energies. The second window is positioned substantially parallel and opposite to the first window defining a first volume therebetween. The first window and the second window are separated by a distance that is sufficiently small such that an electron beam that enters from the first window can propagate through the first sub-chamber and exit from the second window. The at least one second sub-chamber is in fluid communication with the first sub-chamber and has a second volume that is greater than the first volume of the first sub-chamber.

Claims

1. A microfluidic chamber comprising:

(a) a first sub-chamber comprising a first window and a second window that is positioned substantially parallel and opposite to the first window defining a first volume therebetween;

wherein each of the first window and the second window is transparent to electrons of certain energies, and the first window and the second window are separated by a distance such that an electron beam that enters from the first window can propagate through the first sub-chamber and exit from the second window; and

(b) at least one second sub-chamber that is in fluid communication with the first sub-chamber, wherein the at least one second sub-chamber has a second volume that is greater than the first volume of the first sub-chamber.

2. The microfluidic chamber of claim 1, wherein the second volume of the at least one second sub-chamber is dimensioned to allow at least one living cell to be placed therein.

3. The microfluidic chamber of claim 1, wherein the distance between the first window and the second window is smaller than about 10 microns.

4. The microfluidic chamber of claim 3, wherein the distance between the first window and the second window is smaller than about 1 micron.

5. The microfluidic chamber of claim 1, wherein the first sub-chamber and the at least one second sub-chamber are formed between two microchips separated by a spacer.

6. The microfluidic chamber of claim 5, wherein each of the two microchips is made of silicon.

7. The microfluidic chamber of claim 1, wherein each of the first window and the second window is made of silicon nitride.

8. A method of imaging a living cell, comprising the steps of:

(a) placing the cell in a microfluidic chamber formed between two microchips separated by a spacer, the microfluidic chamber comprising;

(i) a first sub-chamber comprising a first window and a second window that is positioned substantially parallel and opposite to the first window defining a first volume therebetween;

wherein each of the first window and the second window is transparent to electrons of certain energies, and the first window and the second window are separated by a distance such that an electron beam that enters from the first window can propagate through the first sub-chamber and exit from the second window; and

(ii) at least one second sub-chamber that is in fluid communication with the first sub-chamber, wherein the at least one second sub-chamber has a second volume that is greater than the first volume of the first sub-chamber;

wherein a larger portion of the cell occupies the at least one second sub-chamber and a smaller portion of the cell occupies the first sub-chamber;

(b) introducing the microfluidic chamber with the cell into a transmission electron microscope (TEM) such that an electron beam can enter the microfluidic chamber through the first window and exit the microfluidic chamber through the second window;

(c) exposing the cell to an electron beam with an electron dosage; and

(d) recording an image of the cell formed by the electron beam.

9. The method of claim 8, wherein the electron dosage is sufficiently low such that the electron beam does not cause structural damage to the cell on a scale larger than the resolution.

10. The method of claim 8, wherein the microfluidic chamber is vacuum sealed such that there is no fluid communication between the microfluidic chamber and a vacuum in the TEM.

11. The method of claim 8, wherein the microfluidic chamber is in fluid communication with an external flow system.

12. The method of claim 11, wherein the external flow system includes means for exchanging fluid with the microfluidic chamber.

13. The method of claim 12, wherein the external flow system includes means for providing nutrients to the cell in the microfluidic chamber.

14. The method of claim 12, wherein the external flow system includes means for injecting chemicals into the microfluidic chamber.

15. The method of claim 12, wherein the external flow system includes means for maintaining a chemical gradient in the microfluidic chamber.

16. The method of claim 11, wherein the external flow system includes means for controlling the temperature of the cell.

17. The method of claim 16, wherein the controlling means includes a thermocouple, a resistive heating wire, a thermoelectric heating element, or a combination thereof.

18. The method of claim 8 further comprising the step of, prior to the exposing step, introducing a plurality of label molecules into the cell for protein identification.

19. The method of claim 18, wherein the introducing step is performed via one of the following methods or a combination thereof:

i) endocytosis;

ii) direct uptake through the cell membrane;

iii) micro-injection;

iv) modification of a DNA of the cell to express proteins enclosing metal atoms; and

v) incubation with a ligand connected to a label for specific binding to a certain receptor, or other molecule.

20. The method of claim 8, wherein the TEM is operated in a phase contrast mode or a scatter contrast mode.

21. The method of claim 8, wherein the TEM is operated in a scanning mode, and contrast.

22. The method of claim 21 further comprising the step of, prior to the exposing step, introducing a plurality of high-Z atoms into the cell.

23. The method of claim 22, wherein the plurality of high-Z atoms comprises gold atoms, or constituents of a fluorescent quantum dot.

24. The method of claim 8 further comprising the steps of, at a time interval after the recording step:

(a) exposing the cell to a second electron beam with a second electron dosage; and

(b) recording a second image of the cell formed by the second electron beam, thereby obtaining dynamic information about the cell.

25. The method of claim 24, wherein the second electron dosage is sufficiently low such that the second electron beam does not cause damage to the cell.

26. The method of claim 8 further comprising the steps of, after the placing step and prior to the exposing step, or after the recording step:

(a) exposing the cell to a light beam; and

(b) recording a light microscopy image of the cell formed by the light beam.

27. The method of claim 26, wherein the light beam has an intensity that is sufficiently low such that the light beam does not cause structural damage to the cell on a scale larger than the resolution.

28. The method of claim 8, wherein the cell is exposed to the electron beam for a time duration less than about 1 microsecond.

29. The method of claim 28 further comprising the steps of, after the recording step:

(a) exposing the cell to a second electron beam for a second time duration that is less than about 1 microsecond; and

(b) recording a second image of the cell formed by the second electron beam, thereby obtaining dynamic information about the cell.

30. The method of claim 8 further comprising the step of, after the placing step and prior to the introducing step, freezing the cell.

31. The method of claim 30, wherein the TEM is operated as a cryo-TEM.

32. The method of claim 8 further comprising the step of, after the placing step and prior to the introducing step, fixing the cell by injecting a chemical into the microfluidic chamber.

33. The method of claim 8, wherein the placing step comprises the steps of:

(a) seeding the cell in the at least one second sub-chamber; and

(b) inducing the cell to grow into the first sub-chamber using one of the following methods or a combination thereof;

i) natural cell adherence; and

ii) using chemicals.

34. A microfluidic chamber comprising:

(a) a first microchip having a substantially flat first surface and a first opening, the first opening being covered by a first window attached to the first surface, the first microchip further having at least one groove formed on the first surface extending from an edge thereof to an interior portion thereof adjacent to the first opening;

(b) a second microchip having a substantially flat second surface and a second opening, the second opening being covered by a second window attached to the second surface; and

(c) a spacer disposed between the first microchip and the second microchip;

wherein the first surface of the first microchip faces the second surface of the second microchip, and the first opening in the first microchip overlaps the second opening in the second microchip in an area, wherein the overlap area defines a first sub-chamber with a first volume, and the at least one groove formed on the first surface of the first microchip and a corresponding portion of the second surface of the second microchip define at least one second sub-chamber with a second volume, wherein the at least one second sub-chamber is in fluid communication with the first sub-chamber, and the second volume of the at least one second sub-chamber is greater than the first volume of the first sub-chamber; and

wherein each of the first window and the second window is transparent to electrons of certain energies, and the spacer has a thickness such that an electron beam that enters from the first window can propagate through the first sub-chamber and exit from the second window.

35. The microfluidic chamber of claim 34, wherein the second volume of the at least one second sub-chamber is dimensioned to allow at least one living cell to be placed therein.

36. The microfluidic chamber of claim 34, wherein the thickness of the spacer is less than about 10 microns, and the at least one groove formed on the first surface of the first microchip has a depth greater than about 50 microns and a width greater than about 50 microns.

37. The microfluidic chamber of claim 36, wherein the thickness of the spacer is less than about 1 micron.

38. The microfluidic chamber of claim 34, wherein each of the first window and the second window is made of silicon nitride.

39. The microfluidic chamber of claim 38, wherein each of the first window and the second window has a thickness of about 50 nm.

40. The microfluidic chamber of claim 34, wherein each of the first microchip and the second microchip is made of silicon.

41. The microfluidic chamber of claim 34, wherein each of the first microchip and the second microchip is made of glass, carbon, metal, or a combination thereof.