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A switchable solvatochromic fluorescent dyad can be used to map ordering of lipids in vesicle membranes at a resolution better than the diffraction limit. Combining a Nile Red fluorophore with a photochromic spironaphthoxazine quencher allows the fluorescence to be controlled using visible light, via photoswitching and FRET quenching. Synthetic lipid vesicles of varying composition were imaged with an average 2.5-fold resolution enhancement, compared to the confocal images. Ratiometric detection was used to probe the membrane polarity, and domains of different lipid ordering were distinguished within the same membrane., (© 2023 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image-with high accuracy-minute differences in the lipid packing of model and cellular membranes., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The performance of a method is assessed which allows for the spatiotemporal tracking of single dye-labeled molecules during two-dimensional (2D) diffusional transits through the focal area of a modified confocal microscope. In addition to facilitating the observation of molecular diffusion paths at the shot-noise limit of bright organic emitters with spatial and temporal precisions of ∼10-20 nm and <0.5 ms, respectively, the direct access to the complete stream of detected photons is beneficial for characterizing nanoscale details such as transient pausing (binding). We discuss technical aspects of this approach, along with results from its application to measuring lipid membrane dynamics in live mammalian cells. Presented topics include a discussion of the advantages of the single-photon collection mode and instrument as well as computational considerations for the localization process. A proof-of-principle experiment shows that optical nanoscopy by stochastic single-molecule switching and position readout could be implementable in parallel with such fast molecular tracking. This would allow direct access to contextual imaging data of local cytoskeletal structural elements or localized longer-lived protein assemblies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Experiments based on fluorescence detection are limited by the population of the fluorescence marker's long-lived dark triplet state, leading to pronounced photobleaching reactions and blinking which reduces the average fluorescence signal obtained per time interval. By irradiation with a second, red-shifted laser line, we initiate reverse intersystem crossing (ReISC) which enhances the fluorescence signal of common fluorophores up to a factor of 14. The reverse intersystem crossing from the triplet state back to the singlet system is achieved by photoexcitation to higher-excited triplet states, which are, however, prone to photobleaching. We gain insights into the competing pathways of ReISC and photobleaching. The relative efficiencies of these two pathways and the triplet lifetime determine the achievable fluorescence enhancement, which varies strongly with the choice of dye, excitation irradiance and wavelength, and with environmental conditions. The study of ReISC not only results in a better understanding of a fluorescent label's photophysics, but the method is a possible approach to optimize fluorescence emission in experiments, where signal strength is a critical parameter.

During the last decade far-field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous-wave laser beam. This bright marker enables strict control of on/off switching and provides single-molecule localization precision down to 15 nm in the focal plane. Two-photon induced nonlinear photoswitching of this marker with continuous-wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost-effective fluorescence nanoscopy with noninvasive optical sectioning.

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.