Imaging Single Lipid Molecules in Living Cells Using Two Photon Excitation

Abstract

A Two Photon Laser Scanning Microscope was used for detecting single Cy3-labeled lipids on the plasma membrane of living human coronary artery smooth muscle cells. For Two-Photon-Excitation of Cy3 the fs-laser system was tuned to 700nm [1]. Figure A shows a fluorescence image of blank HASM cells taken with a mean power of 6mW at the sample and a dwell time of 50ms per pixel. The spots around the nucleus originate from autofluorescence of mitochondria. After taking this blank image the cells were incubated with a vesicle solution of POPC/DMPE-Cy3 at a molar ratio of 103.Lipid exchange between the vesicles and the cells resultedin a concentration of Cy3-labeled lipids of ∼1/50 μm2 on the cell membrane [2]. The vesicles were removed out of the measurement chamber by flushing with PBS buffer. For single molecule detection experiments a scanning region with almost no autofluorescence was chosen as indicated in Figure A. A sequence of images as shown Figure B was taken with a dwell time of 50ms per pixel. The fluorescence peaks represent single Cy3-labeled lipids diffusing on the cell membrane. Imaging the diffusion of single fluorescence labeled lipids by TPE has been accomplished for an artificial lipid membrane [3]. Here we show that single molecule detection with TPE is also possible on cells in vivo. This opens the perspective to utilize the advantages of Two Photon Excitation, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multicolor experiments, for single molecule imaging in living biological cells.