Supercontinuum Stimulated Emission Depletion Fluorescence Lifetime Imaging

Abstract

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 theX 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. ABSTRACT: 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.

Photon Counting Data Analysis: Application of the Maximum Likelihood and Related Methods for the Determination of Lifetimes in Mixtures of Rose Bengal and Rhodamine B

“…Depending on whether the fluorescence decay is described by two or one decaying exponentials, the expression for pj is given by either equation (4) or equation (6). The probability that the photon is not detected (failure) in the jth bin is given by = 1 − .…”

“…[3][4][5][6] Typically, in a TCSPC experiment, a fluorescence lifetime is determined by acquiring a histogram of arrival time differences between an excitation pulse and the pulse resulting from a detected photon. As we have noted, 3,4 when a histogram of sufficient quality cannot be obtained to provide a good fit by means of minimizing the residuals (RM) between the experimental data and a given functional form, the maximum likelihood (ML) technique is particularly effective, namely when the total number of counts is very low.…”

Review of recent developments in stimulated emission depletion microscopy: applications on cell imaging

“…Since the first introduction, the strengths of such microscopes in resolving nanoscopic structures in live and fixed cells have been demonstrated by several research groups. [36][37][38][39][40][41][42][43] The p-STED microscopy is widely used in studying fixed biological cells. A particularly interesting demonstration of p-STED microscopy resolved the organization of the protein subunits of mitofilin, MINOS1, and CHCHD3 residing in mitochondrial-inner-membrane-organization-system (MINOS) in different chemically fixed cell types.…”

“…Such a setup has been used by other groups in STED fluorescence lifetime imaging. 36,43 Another approach that uses discrete multicolor-stimulated Raman scattering light as the depletion source has been applied in STED microscopy. 94 The output of a Q-switched microchip laser at 532 nm is sent through a polarization-maintaining single-mode fiber, which emits seven discrete laser lines in the range of 532 to 620 nm.…”

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…FLIM has also been combined with other techniques, such as fluorescence correlation spectroscopy (FCS) (Becker et al, 2006;Breusegem et al, 2006;Nguyen et al, 2012), scanning near-field optical microscopy (SNOM) (Kwak et al, 2001), atomic force microscopy (AFM) (Micic et al, 2004), selective plane illumination microscopy (Greger et al, 2011), fluorescence recovery after photobleaching (FRAP) (Levitt et al, 2011a;Roberti et al, 2011;Vitali et al, 2010), total internal reflection (TIRF) microscopy (Bruns et al, 2008;Devauges et al, 2012), STED (Auksorius et al, 2008;Bückers et al, 2011;Lesoine et al, 2012;Lin et al, 2013), coherent anti-Stokes Raman scattering (CARS) (Slepkov et al, 2011) and tomography (McGinty et al, 2011).…”