Tracking movements of lipids and Thy1 molecules in the plasmalemma of living fibroblasts by fluorescence video microscopy with nanometer scale precision

The lateral diffusion of 100 nm fluorescent latex microspheres (FS) bound to either N-biotinylphosphatidyl-ethanolamine or the glycosylphosphatidylinositol-linked protein Thy1 were monitored in the plasmalemma of primary rat fibroblasts by single particle tracking of FS centroids from digital fluorescence micrographs. A silicon intensified target camera was found to be superior to slow scan cooled CCD and intensified interline transfer CCD cameras for monitoring lateral diffusion of rapidly moving FS with nanometer level precision. To estimate the maximum tracking precision, a 4 sec-sequence comprising 120 images of FS fixed to a cover glass was obtained. The mean distance of the centroids from the origin was 7.5 ± 0.4 nm, and no centroids were beyond 16 nm from the origin. The SIT camera was then used to track FS attached to lipids and Thy1 molecules on the surface of fibroblasts. The lateral diffusion of lipid-bound FS was unconstrained, and the ensemble averaged diffusion coefficient was 0.80 × 10−9 cm2/sec. Thy1-bound FS existed in two mobility populations, both of which demonstrated constrained mobility. The rapidly moving population, comprising 61% of the total, had an ensemble diffusion coefficient of 6.1 × 10−10 cm2/sec, and appeared to be restricted to domains with a mean length of about 700 nm. The slowly moving population, comprising about 39% of the total, had a diffusion coefficient of 5.7 × 10−12 cm2/sec. These results demonstrate that nanovid can be extended to the realm of fluorescence microscopy and support previous studies indicating that while the lateral mobilities of at least some lipids are not constrained to small domains by barriers to lateral diffusion in the fibroblast plasmalemma, a peripheral membrane protein which is bound only by a lipid anchor can be prevented from diffusing freely.