Aleš Benda

Fluorescence Lifetime Correlation Spectroscopy

This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The usefulness of the method is demonstrated on simple FCS experiments. The method makes possible to separate the autocorrelation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing.

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

GFP-tagged snRNP components reveal the dynamics and rate for spliceosome assembly in vivo.

Morphology and Carrier Extraction Study of Organic–Inorganic Metal Halide Perovskite by One- and Two-Photon Fluorescence Microscopy

The past two years have seen the uniquely rapid emergence of a new class of solar-cell-based on mixed organic-inorganic halide perovskite. In this work, we demonstrate a promising technique for studying the morphology of perovskite and its impact on carrier extraction by carrier transport layer using one-photon and two-photon fluorescence imaging in conjunction with time-resolved photoluminescence. This technique is not only effective in separating surface and bulk effects but it also allows the determination of lifetimes in localized regions and local carrier extraction efficiency. The difference in sensitivities of transport materials to grain boundaries and film uniformity is highlighted in this study. It is shown that the PCBM fabricated in this work is more sensitive to film nonuniformity, whereas spiro-OMeTAD is more sensitive to grain boundaries in terms of effective carrier extraction.

Spectral analysis of doxorubicin accumulation and the indirect quantification of its DNA intercalation.

There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.

TCSPC upgrade of a confocal FCS microscope

We extended the measurement capabilities of the Carl Zeiss ConfoCor 1 FCS microscope by (a) using pulsed picosecond diode lasers instead of a continuous wave (CW) laser excitation, (b) introducing a fast single photon avalanche diode detector, and (c) exploiting the capabilities of the PicoQuant TimeHarp 200 board. When the time-tagged time-resolved (TTTR) mode of the TimeHarp is utilized, the complete fluorescence dynamics are recorded. That is, the time-evolution of the fluctuations and the fluorescence decay kinetics are captured simultaneously. Recording individual photon events (without on-the-fly data reduction like in hardware correlators) preserves the full information content of the measurement for virtually unlimited data analysis tasks and provides a much more detailed view of processes happening in the detection volume. For example, autocorrelation functions of dyes in a mixture can be separated and/or their cross-correlation can be investigated. These virtual two-channel measurements are perf...

Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector after pulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined.

Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations

Although there were experimental indications that phospholipid bilayers hydrated with D(2)O express different biophysical properties compared with hydration by ordinary H(2)O, a molecular concept for this behavior difference was only recently proposed by a molecular dynamics simulations study [T. Róg et al., J. Phys. Chem. B, 2009, 113, 2378-2387]. Here we attempt to verify those theoretical predictions by fluorescence measurements on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Specifically, we determine the water isotope effect on headgroup hydration and mobility, lateral lipid diffusion and lipid backbone packing. Time-dependent fluorescence shift experiments show significantly slower dynamics and lower hydration of the headgroup region for a bilayer hydrated with D(2)O, an observation in good agreement with the calculated predicted differences in duration of lipid-lipid and lipid-water bridges and extent of water penetration into the bilayer, respectively. The water isotope effect on the lipid order parameter of the bilayer core (measured by fluorescence anisotropy) and lateral diffusion of lipid molecules (determined by two-focus fluorescence correlation spectroscopy) is close to the experimental errors of the experiments, however also refers to slightly more rigid organization of phospholipid bilayers in heavy water. This study confirms the view that the water isotope effect can be particularly found in time-resolved physicochemical properties of the membrane. Together with the simulations our experiments provide a comprehensive, molecular view on the effect of D(2)O on phospholipid bilayers.

Optical Saturation as a Versatile Tool to Enhance Resolution in Confocal Microscopy

One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbes diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.

In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer

Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.

Fluorescence Lifetime Tuning—A Novel Approach to Study Flip-Flop Kinetics in Supported Phospholipid Bilayers

In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.