U.S. Department of Energy, Ames Laboratory , Ames, Iowa, United States; Department of Chemistry, Iowa State University, Ames, Iowa, United States.
J Phys Chem B. 2012 Jul 12;116(27):7821-6. doi: 10.1021/jp303912p. Epub 2012 Jun 27.
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 ± 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 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 ± 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.
利用时间相关单光子计数（TCSPC）检测，实现了超连续谱（SC）受激辐射耗散（STED）荧光寿命成像。通过对单分散 40nm 荧光珠成像来测量所开发的 STED 仪器的空间分辨率，然后确定其半峰全宽（FWHM），X 和 Y 坐标的分辨率分别为 36±9nm 和 40±10nm。共聚焦显微镜测量的相同珠子的分辨率为 450±50nm 和 430±30nm，这大于由于未充满显微镜物镜而产生的光的衍射极限。为了实现所声称的 STED 空间分辨率，有必要对物镜进行欠填充并对信号进行时间门控。通过共聚焦或 STED 寿命成像，对荧光珠进行了相同的荧光寿命（2.0±0.1ns）的测量。该仪器已应用于研究培养细胞中小于光的衍射极限的尺寸的、用 Alexa Fluor 594-鬼笔环肽标记的富含 F-肌动蛋白的突起。通过 STED 寿命成像测量，富含肌动蛋白的突起的荧光寿命范围为 2.2 至 2.9ns。
