Supercontinuum Stimulated Emission Depletion Fluorescence Lifetime Imaging

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• 作者：Michael D. Lesoine ; Sayantan Bose ; Jacob W. Petrich ; Emily A. Smith
• 刊名：The Journal of Physical Chemistry B
• 出版年：2012
• 出版时间：July 12, 2012
• 年：2012
• 卷：116
• 期：27
• 页码：7821-7826
• 全文大小：382K
• 年卷期：v.116,no.27(July 12, 2012)
• ISSN：1520-5207

文摘

Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by 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 卤 9 and 40 卤 10 nm in the X and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 卤 50 and 430 卤 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 卤 0.1 ns) was measured for the fluorescent beads by 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.