Super-continuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their FWHM, and was 36 � 9 and 40 � 10 nm in the X and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 � 50 and 430 � 30 nm, which is larger than the diffraction limit of light due to under filling the microscope objective. Under filling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 � 0.1 ns) was measured for the fluorescent beads using confocal or STED lifetime imaging. 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 cells. Fluorescence lifetimes of the actin-rich projections range from 2.2 to 2.9 ns as measured by STED lifetime imaging. Figure 1. <img src='https://acs-archive-assets2.s3.amazonaws.com/239nm/uploads/8946_clip_image002.jpg' alt='' />Lesoine, M. D.; Bose, S.; Petrich, J. W.; Smith, E. A. Supercontiuum Stimulated Emission Depletion Fluorescence Imaging. J. Phys. Chem. B. 2012 , 116, 7821-7826.Lesoine, M.D.; Bhattacharjee, U.; Guo, Y.; Vela, J.; Petrich, J.W.; Smith, E.A. Subdiffraction, Luminescence-Depletion Imaging of Isolated, Giant, CdSe/CdS Nanocrystal Quantum Dots. J. Phys. Chem. C., 2013 , 117 (7), 3662-3667.

Development and application of a stimulated emission depletion (STED) microscope

246th National Meeting (2013)

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