Photobleaching reduced fluorescence correlation spectroscopy

A dual beam excitation-depletion pulse technique is proposed for photobleaching reduced fluorescence correlation spectroscopy (FCS). Excitation pulse promote the molecules to the excited singlet state (S-1), a fraction of that population goes to energetically favorable metastable triplet state (T-1) due to strong intersystem crossing. The depletion pulse followed by excitation pulse instantaneously depletes the triplet states thereby recycling the bleached molecules back to the ground state (S-0). FCS study on diffusing Fluorescein and Rh6G molecules show more than 95% reduction in triplet state population and the associated photobleaching. (c) 2010 American Institute of Physics.

Coexistence of fast photodarkening and slow photobleaching in Ge19As21Se60 thin films

We experimentally demonstrate the coexistence of two opposite photo-effects, viz. fast photodarkening (PD) and slow photobleaching (PB) in Ge19As21Se60 thin films, when illuminated with a laser of wavelength 671 nm. PD appears to begin instantaneously upon light illumination and saturates in tens of seconds. By comparison, PB is a slower process that starts only after PD has saturated. Both PD and PB follow stretched exponetial dependence on time. Modeling of overall change as a linear sum of two contributions suggests that the changes in As and Ge parts of glass network respond to light effectively indepndent of each other. (C) 2012 Optical Society of America

Crossover from photodarkening to photobleaching in a-GexSe100-x thin films

In this Letter, we present the interesting results of photodarkening (PD), transition toward photostability, and a slow crossover from PD to photobleaching when composition of the chalcogenide glassy thin film changes from Ge-deficient to rich. A subsequent Raman analysis on these as-prepared and irradiated samples provide the direct evidence of photoinduced structural rearrangement, i.e., photocrystallization of Se and the removal of edge-sharing GeSe4 tetrahedra. Further, our experimental results clearly demonstrate that light-induced effects can be effectively controlled by choosing the right composition and provide valuable information on synthesizing photostable/sensitive glasses.

Observation of photobleaching and intensity dependent kinetics in Ge22As22Se56 thin films under sub-bandgap light illumination

We experimentally demonstrate photobleaching (PB) in Ge22As22Se56 thin films, when illuminated with a diode pumped solid state laser (DPSSL) of wavelength 671 nm, which is far below the optical bandgap of the sample. Interestingly, we found that PB is a slow process and occurs even at moderate pump beam intensity of 0.2 W/cm(2), however the kinetics remain rather different.

Generation of extended light-sheets for single and multi-photon fluorescence microscopy

We theoretically propose and computationally demonstrate the generation of extended light-sheet for fluorescence microscopy. This is made possible by the introduction of a specially designed double-window spatial filter that allows the light to pass through the periphery and center of a cylindrical lens. When illuminated with a plane wave, the proposed filter results in an extended depth-of-focus along with side-lobes which are due to other interferences in the transverse focal plane. Computational studies show a maximum extension of light-sheet by 3.38 times for single photon excitation and 3.68 times for multiphoton excitation as compared to state-of-art single plane illumination microscopy system. This technique may facilitate the study of large biological specimens (such as Zebrafish embryo and tissue) with high spatial resolution and reduced photobleaching. (C) 2013 AIP Publishing LLC.

High-resolution restoration of 3D structures from widefield images with extreme low signal-to-noise-ratio

Four-dimensional fluorescence microscopy-which records 3D image information as a function of time-provides an unbiased way of tracking dynamic behavior of subcellular components in living samples and capturing key events in complex macromolecular processes. Unfortunately, the combination of phototoxicity and photobleaching can severely limit the density or duration of sampling, thereby limiting the biological information that can be obtained. Although widefield microscopy provides a very light-efficient way of imaging, obtaining high-quality reconstructions requires deconvolution to remove optical aberrations. Unfortunately, most deconvolution methods perform very poorly at low signal-to-noise ratios, thereby requiring moderate photon doses to obtain acceptable resolution. We present a unique deconvolution method that combines an entropy-based regularization function with kernels that can exploit general spatial characteristics of the fluorescence image to push the required dose to extreme low levels, resulting in an enabling technology for high-resolution in vivo biological imaging.

Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules

Plasmon rulers, consisting of pairs of gold nanoparticles, allow single-molecule analysis without photobleaching or blinking; however, current plasmon rulers are irreversible, restricting detection to only single events. Here, we present a reversible plasmon ruler, comprised of coupled gold nanoparticles linked by a single aptamer, capable of binding individual secreted molecules with high specificity. We show that the binding of target secreted molecules to the reversible plasmon ruler is characterized by single-molecule sensitivity, high specificity, and reversibility. Such reversible plasmon rulers should enable dynamic and adaptive live-cell measurement of secreted single molecules in their local microenvironment.