Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording

Abstract

In this work, a concept is described for how the kinetics of photoinduced, transient, long-lived, nonfluorescent or weakly fluorescent states of fluorophore marker molecules can be extracted from the time-averaged fluorescence by using time-modulated excitation. The concept exploits the characteristic variation of the population of these states with the modulation parameters of the excitation and thereby circumvents the need for time resolution in the fluorescence detection. It combines the single-molecule sensitivity of fluorescence detection with the remarkable environmental responsiveness obtainable from long-lived transient states, yet does not in itself impose any constraints on the concentration or the fluorescence brightness of the sample molecules that can be measured. Modulation of the excitation can be performed by variation of the intensity of a stationary excitation beam in time or by repeated translations of a CW excitation beam with respect to the sample. As a first experimental verification of the approach, we have shown how the triplet-state parameters of the fluorophore rhodamine 6G in different aqueous environments can be extracted. We demonstrate that the concept is fully compatible with low time-resolution detection by a CCD camera. The concept opens for automated transient-state monitoring or imaging on a massively parallel scale and for high-throughput biomolecular screening as well as for more fundamental biomolecular studies. The concept should also be applicable to the monitoring of a range of other photoinduced nonfluorescent or weakly fluorescent transient states, from which subtle changes in the immediate microenvironment of the fluorophore marker molecules can be detected.

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

“…The resulting decreased molecular brightness of A is not an issue for the TRAST method as it can be compensated for by using higher concentrations or longer acquisition times. 14 The results also predict that the technique is applicable to imaging using a standard laser scanning microscope or a wide-field microscope equipped with a time-modulated excitation. As a proof of principle, we have performed TRAST measurements in a one point modus.…”

“…[14][15][16] In TRAST, the excitation intensity is modulated in an on-off fashion while recording the time-averaged fluorescence response. During each excitation pulse, the triplet and radical state populations build up over time, leading to a decrease in fluorescence.…”

Trans–Cis Isomerization of Lipophilic Dyes Probing Membrane Microviscosity in Biological Membranes and in Live Cells

“…In a TRAST measurement, applying low duty cycle excitation pulse trains with different , the isomerization parameters of MC540 can then in principle be determined from the dependence of 〈 〉( ), as has been shown for several other long-lived, photo-induced transient states 23,25,26 . For MC540, this -dependence is determined by the time dependence of the probability of the fluorophore to be in a trans state ( ), which can be described by the model of equation 1, as further outlined in the SI.…”

“…By mapping the position of the excitation beam onto the camera and using the determined radii, the absorption cross-section of the dye and the measured power after the objective could be determined, and an effective excitation rate could be assigned to each pixel on the camera (Equations 12-13). 23,26 Similarly, for confocal measurements the beam widths were calculated by applying a 2D Gaussian distribution fit to the each image of the sample, yielding 1 2 ⁄ radii of 0,9μm/0,5μm and 1,6μm/1,15μm in ethanol and in the vesicle solution respectively. The non-zero extinction factor of the AOM (approximately 0.1%) was accounted for by subtracting an image recorded with the AOM turned off from the other images.…”

“…This puts a limit to the application range and throughput of FCS measurements, and makes parallel readouts and imaging difficult. To overcome these limitations, we have introduced an approach, so-called transient state (TRAST) imaging 23,24 , where the fluorescent sample is subject to a modulated excitation. Depending on the excitation pulse train characteristics (e.g.…”