MRT letter: High speed scanning has the potential to increase fluorescence yield and to reduce photobleaching

Abstract

True confocal microscopy requires point-shaped illumination and detection. To generate an image, a diffraction limited spot is moved over the sample. Single spot scanning has suffered in the past from low image rates; a solution is the employment of very fast scanning devices (resonant scanners) for x-movement. In the process of introducing resonant scanning devices, it was found that both signal yield is improved and bleaching is decreased-in contrary to the assumed performance. This article will show by a simple and well understood model a straightforward explanation for the potential increase of signal yield and decrease in photobleaching. The time that is ruling the dose-rate effects is the effective time; a fluorochrome is illuminated. This time depends on the diameter of the spot that is moved over the sample and the speed at which the spot moves. In essence, the scan process causes a pulsed illumination of the fluorochromes. Various schemes of pulsed illumination are simulated with a fluorescence model. The model includes a dark state, where fluorochromes will exit the fluorescence process and slowly decay back into the ground state. Upon splitting a single dose into two pulses separated by a dark time-reflecting an increased scan speed-the amount of fluorescence emission is increased and bleaching is reduced. These results show a potential increase of fluorescence and a lower photobleaching upon higher scan speed. As illumination during the bleach-phase in a FRAP-experiment is similar to a light pulse, the findings also suggest to critically consider the very beginning of fluorescence recovery in terms of triplet relaxation process that potentially could falsify the measurements.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in <em>Arabidopsis</em>

“…While this shorter procedure proved functional for several immunostaining [22][23][24] , we found that it attenuates the robustness of the staining across samples, antibody and batch of cell wall digestion enzyme mix (see below). Furthermore we were not successful for FISH hybridization with this short procedure.…”

“…In addition, we successfully used this protocol to quantitatively analyze chromatin dynamics by immunostaining in megaspore mother cells, functional megaspore, developing female gametophytes and early embryo 21,[22][23][24] . In Figure 5, we show representative results of whole-mount immunostaining on Arabidopsis ovules.…”

Live-cell imaging

“…The period and duty cycle of the outer pulse is highly variable. The units of the outer pulse is in 1/10 000 s/unit, and the PwM intensity varies between 0-800. reduce photo-toxicity 38,[40][41][42] while maintaining image quality. Compared with continuous wave (CW) illumination, shorter pulse widths reduced illumination and the time lag between two pulses allows all excited molecules to non-radiatively relax to the ground states via non-radiative and intermolecular (i.e., conduction and convection) as well as radiative (i.e., flourescence and phosphorecence) de-excitation processes.…”

Ultrafast photon counting applied to resonant scanning STED microscopy

“…Recent developments in STED microscopy include: 1) the usage of resonant scanning mirrors that allow fast scanning (Moneron et al 2010;Westphal et al 2007) and thus, decrease the rate of fluorophore photobleaching due to triplet states buildup (Borlinghaus 2006;Tsien and Bacskai 1995); and 2) the implementation of time-gated detection with low-power continuous wave (CW) depletion lasers that reach a resolution of ~60–70 nm in biological samples (Vicidomini et al 2011;Vicidomini et al 2013). Yet, the speed of the photon counting systems limits the linear scanning speed that can be used with resonant mirrors in STED microscopes.…”