Multi-target spectrally resolved fluorescence lifetime imaging microscopy.

We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

Integration of a superconducting nanowire single-photon detector into a confocal microscope for time-resolved photoluminescence (TRPL)-mapping: Sensitivity and time resolution.

This report highlights the combination of the MicroTime 100 upright confocal fluorescence lifetime microscope with a Single Quantum Eos Superconducting Nanowire Single-Photon Detector (SNSPD) system as a powerful tool for photophysical research and applications. We focus on an application in materials science, photoluminescence imaging, and lifetime characterization of Cu(InGa)Se2 (CIGS) devices intended for solar cells. We demonstrate improved sensitivity, signal-to-noise ratio, and time-resolution in combination with confocal spatial resolution in the near-infrared (NIR) range, specifically in the 1000-1300 nm range. The MicroTime 100-Single Quantum Eos system shows two orders of magnitude higher signal-to-noise ratio for CIGS devices' photoluminescence imaging compared to a standard NIR-photomultiplier tube (NIR-PMT) and a three-fold improvement in time resolution, which is now limited by the laser pulse width. Our results demonstrate the advantages in terms of image quality and time resolution of SNSPDs technology for imaging in materials science.

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing.

Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

Multi-target immunofluorescence by separation of antibody cross-labelling via spectral-FLIM-FRET.

In biomedical research, indirect immunofluorescence labelling by use of primary and secondary antibodies is central for revealing the spatial distribution of multiple cellular antigens. However, labelling is regularly restricted to few antigens since species variation of primary and corresponding secondary antibodies is limited bearing the risk of unspecific cross-labelling. Here, we introduce a novel microscopic procedure for leveraging undesirable cross-labelling effects among secondary antibodies thereby increasing the number of fluorophore channels. Under cross-labelling conditions, commonly used fluorophores change chemical-physical properties by 'Förster resonance energy transfer' leading to defined changes in spectral emission and lifetime decay. By use of spectral fluorescence lifetime imaging and pattern-matching, we demonstrate precise separation of cross-labelled cellular antigens where conventional imaging completely fails. Consequently, this undesired effect serves for an innovative imaging procedure to separate critical antigens where antibody species variation is limited and allows for multi-target labelling by attribution of new fluorophore cross-labelling channels.

Resolving dynamics and function of transient states in single enzyme molecules.

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.

Enthalpy-driven RNA folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction.

RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop-receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21-47 degrees C), from which a van't Hoff analysis yields the enthalpy (DeltaH degrees) and entropy (DeltaS degrees) of docking. Tetraloop-receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl(2) and 100 mM NaCl, with excellent agreement between immobilized (DeltaH degrees = -17.4 +/- 1.6 kcal/mol, and DeltaS degrees = -56.2 +/- 5.4 cal mol(-1) K(-1)) and freely diffusing (DeltaH degrees = -17.2 +/- 1.6 kcal/mol, and DeltaS degrees = -55.9 +/- 5.2 cal mol(-1) K(-1)) species. Kinetic heterogeneity in the tetraloop-receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop-receptor interaction can account for approximately 60% of the DeltaH degrees and DeltaS degrees of P4-P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop-receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions.

Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy.

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

Spectral identification of specific photophysics of cy5 by means of ensemble and single molecule measurements.

The triplet-state characteristics of the Cy5 molecule related to trans-cis isomerization are investigated by means of ensemble and single molecule measurements. Cy5 has been used frequently in the past 10 years in single molecule spectroscopic applications, e.g., as a probe or fluorescence resonance energy transfer acceptor in large biomolecules. However, the unknown spectral properties of the triplet state and the lack of knowledge on the photoisomerization do not allow us to interpret precisely the unexpected single molecule behaviors. This limits the application of Cy5. The laser photolysis experiments demonstrate that the trans triplet state of Cy5 absorbs about 625 nm, the cis ground state absorbs about 690 nm, and the cis triplet state also absorbs about 690 nm. In other words, the T1-Tn absorptions largely overlap the ground-state absorptions for both trans and cis isomers, respectively. Furthermore, the observation of the cis triplet state indicates an important isomerization pathway from the trans-S1 state to the cis-T1 state upon excitation. The detailed spectra presented in this article let us clearly interpret the exact mechanisms responsible for several important and unexpected photophysical behaviors of single Cy5 molecules such as reverse intersystem crossing (RISC), the observation of dim states with a lower emission intensity and slightly red-shifted fluorescence, and unusual energy transfer from donor molecules to dark Cy5 molecules acting as acceptors in single molecule fluorescence resonance energy transfer (FRET) measurements. Spectral results show that the dim state in the single molecule fluorescence intensity time traces originated from cis-Cy5 because of a lower excitation rate, resulting from the red-shifted ground-state absorption of cis-Cy5 compared to that of the trans-Cy5.

Direct observation of delayed fluorescence from a remarkable back-isomerization in Cy5.

The direct observations of delayed fluorescence and phosphorescence from the cyanine dye Cy5 are reported. The delayed fluorescence is generated from the S(1) state of trans-Cy5 through a reserve intersystem crossing from the cis-triplet state T(1) to the trans-singlet state S(1) via thermal activation. The lowest cis-triplet state is evidenced to be involved in the formation of the isomer. The back-isomerization from cis-triplet state to trans-singlet state crossing, a remarkably back-isomerization pathway that has not been reported before, plays a significant role in this unusual delayed fluorescence.