We developed a stimulated emission depletion (STED) fluorescence microscope and used it to collect sub-diffraction fluorescence lifetime images of cellular structures. The basic operating concept of STED microscopy is inhibiting the fluorescence at the periphery of a diffraction-limited excitation spot by stimulated emission. Combined with time-correlated single-photon counting detection, fluorescence lifetime images with 40 nm lateral spatial resolution are measured at ambient laboratory conditions. The same average fluorescence lifetime (2.0 � 0.1 nanoseconds) was measured for 40-nm fluorescent beads using confocal or STED lifetime imaging, proving that the STED format does not convolute the measured fluorescence lifetime. Three critical parameters affecting the performance of the system are beam profile, signal gating, and optical alignment. The effects of the number of accumulated photons on the STED fluorescence lifetime images will be discussed. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin rich projections with dimensions smaller than the diffraction limit of light in cultured S2 cells.  Fluorescence lifetimes of the F-actin rich projections range from 2.2 to 2.9 nanoseconds as measured by STED lifetime imaging, which is less than the solution fluorescence lifetime due to quenching within the cell.  Factors that affect the F-actin organization will be discussed.

Sub-diffraction fluorescence lifetime imaging in cultured cells

248th National Meeting (2014)

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