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Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Exciting developments: Switching individual photochromic and fluorescent rhodamine amides enables 3D far‐field optical microscopy with nanoscale resolution, excellent signal‐to‐noise ratio, and fast acquisition times. The rhodamine amides can be switched on using two photons, which enables 3D detailed imaging of thick and densely stained samples (such as 5‐μm silica beads (see image) and living cells) to be constructed.

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We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

We extended the sensitivity of Raman correlation spectroscopy in solution to the single-molecule level by applying surface- and resonance-enhanced Raman scattering (SERRS) combined with time-gated, confocal signal detection. The brightness of the SERRS signal of single Rhodamine 6G molecules adsorbed on a single silver nanoparticle is comparable to fluorescence. Rare event analysis reveals the existence of few particles with simultaneous SERRS and fluorescence signal. The observation of a dynamic exchange between heterogeneous binding sites is supported by the existence of multiple SERRS brightnesses in the signal intensity distribution and by signal fluctuations in the 60 μs time range detected by autocorrelation analysis. Finally, polarization-dependent SERRS autocorrelation curves and single-particle analysis allowed us to measure individual rotational diffusion times and to directly analyze the heterogeneity of the ensemble in solution.

Cellular functions rely on proper actions of organelles such as peroxisomes. These organelles rely on the import of proteins from the cytosol. The peroxisomal import receptor PEX5 takes up target proteins in the cytosol and transports them to the peroxisomal matrix. However, its cytosolic molecular interactions have so far not directly been disclosed. Here, we combined advanced optical microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and STED microscopy with biochemical tools to present a detailed characterization of the cytosolic diffusion and interaction dynamics of PEX5. Among other features, we highlight a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol as well as highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.

Using two-photon excitation (TPE) at 700 nm as well as one-photon excitation (OPE) at 350 nm, we applied confocal fluorescence microscopy to detect single Coumarin-120 molecules in the solvents water and triacetin. To study the behavior of Coumarin-120 under different excitation conditions, fluorescence lifetimes, multichannel scaler traces, and autocorrelation curves have been measured simultaneously. A signal-to-background ratio of 1300 was achieved for TPE due to a very low background level. The detection efficiency of TPE is limited by other competing nonlinear processes, in particular continuum generation in the solvent. The applicable laser intensity for OPE is limited by two-step photolysis of the dye as shown by fluorescence correlation spectroscopy (FCS). The time-resolved fluorescence signals were analyzed by a maximum likelihood estimator to identify the fluorophore through its characteristic fluorescence lifetime. The average fluorescence lifetimes 4.8 ± 1.2 ns in water and 3.3 ± 0.6 ns in triacetin are in good agreement with results obtained from separate measurements at higher concentrations.

AbstractTwo-photon excitation with a mode-locked titanium: sapphire laser at 700 nm and confocal fluorescence microscopy have been used to detect single Coumarin-120 (C-120) molecules. The dye C-120 is quenched by the nucleobases, if coupled to nucleotides, resulting in nucleobase-specific fluorescence lifetimes. This suggests applications in current projects for ultrasenstive DNA characterization.

The efficiency of detecting a single fluorescent coumarin dye molecule in aqueous solution by one‐photon excitation (OPE) at 350 nm as well as by coherent two‐photon excitation (TPE) at 700 nm is studied. The photostability, which is crucial for single molecule detection (SMD), is determined at a low irradiance for various coumarin derivatives using a ‘cell‐bleaching’ method. The yields of photobleaching for these coumarins in aqueous solution are in the order of 10−3 to 10−4. Thus, most of the dyes are sufficiently stable to allow SMD. However, for SMD in a fluorescence microscope a high quasi‐CW irradiance (at least 104 W cm−2) is necessary for efficient OPE by a pulsed, frequency doubled titanium:sapphire laser. Detailed investigations on the dye Coumarin‐120 using fluorescence correlation spectroscopy (FCS), different repetition rates of the laser and transient absorption spectroscopy (TRABS) gave clear evidence that OPE at a high irradiance results in two‐step photolysis via the first electronic excited singlet and triplet state, S1 and T1, producing dye radical ions and solvated electrons. Hence, this additional photobleaching pathway limits the applicable irradiance for OPE. Using coherent TPE for single molecule detection, saturation of the fluorescence was observed for a high quasi‐CW irradiance (108 W cm−2), which may also be caused by photobleaching. Furthermore, TPE is deteriorated by other competing nonlinear processes (e.g. continuum generation in the solvent), which only occur above a threshold irradiance (7 × 107 W cm−2). Nevertheless, TPE allows an efficient detection of single Coumarin‐120 molecules in water. Using a maximum likelihood estimator, we are also able to identify single dye molecules via their characteristic fluorescence lifetime of 4.8 ± 1.2 ns.

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) is introduced. BIFL allows simultaneous registration of fluorescence intensity, lifetime, and anisotropy. It is shown to be well-suited to identify the freely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fluorescent Protein via their characteristic fluorescence anisotropy using a time-resolved analysis. Furthermore, data analysis is discussed and rotational correlation times of single molecules are determined. Applications for multidimensional single-molecule identification are outlined.

Using a confocal epi-illuminated microscope together with a pulsed laser, new applications of the recently developed, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) are introduced. BIFL registers two different types of information on every detected photon with regard to the macroscopic time scale of a measurement and to the fluorescence lifetime. Thus, it is shown to be well suited to identify freely diffusing single dye molecules via their characteristic fluorescence lifetime. This allows for selective counting of dye molecules in an open volume element and opens up the possibility to quantify the relative concentration of the dye molecules, using a recently derived theoretical model, which analyzes the obtained burst size distribution of a sample survey. A closed theory is presented to calculate the probability of a specific dye to cause a fluorescence burst containing a certain number of detected photons. It considers the distribution of the excitation irradiance over the detection volume together with saturation effects of the fluorescence and of the detection electronics, the probability of different transit times through the detection volume, and the probability of multimolecule events. Using BIFL together with selective counting, the concentration of two dyes, Rhodamine B and Rhodamine 6G, in separate solutions and in a mixture were determined. The obtained results are consistent with the applied dye concentrations and with simultaneous measurements by fluorescence correlation spectroscopy (FCS). The introduced method is an appropriate tool for the complete characterization and quantitative analysis of a highly diluted sample in homogeneous assays.