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Single Molecule Microscopy for Living Cells

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• (pdf) - Douglass, A.D. and Vale, R.D. (2005) Single-Molecule Microscopy Reveals Plasma Membrane Microdomains Created by Protein-Protein Networks that Exclude or Trap Signaling Molecules in T Cells. Cell 121: 937-950. (cover).

Single molecule imaging in live cells is preformed using the custom-built TIRF microscope (picture). The angle of the illumination from various laser lines (behind the scope) can be tuned and total internal reflection is easily achieved at the interface of the specimen and the TIRF-illumination objective (1.45 NA) installed in a Zeiss Axiovert 200M inverted fluorescence microscope. The fluorescence signal from the sample under the TIRF illumination is detected by a high sensitive intensified CCD camera (Stanford Photonics Mega 10 S30Z). The system is used to image dynamics of a single GFP molecule in Jurkat T-cell membranes at the rate of 90 frames per second, which revealed the structure and organization of membrane signaling domains of T-cells (Douglass and Vale, 2005).

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