Using Time-Correlated Single Photon Counting (TCSPC) for the purpose of fluorescence lifetime measurements is usually limited in speed due to pile-up. With modern instrumentation this limitation can be lifted significantly but some artefacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remains an issue to be addressed. We propose here a data analysis method correcting for this type of artefact and the resulting systematic errors. It physically models the photon losses due to detector pulse pile-up and incorporates the loss in the decay fit model employed to obtain fluorescence lifetimes and relative amplitudes of the decay components. Comparison of results with and without this correction show a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescense lifetime imaging (FLIM) at very high frame rates.
Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.
Combining stimulated-emission-depletion microscopy with fluorescencecorrelation spectroscopy enables the simultaneous confocal and super-resolution monitoring of molecular diffusion. Cell membranes play an active role in many biological processes and are complex, highly heterogeneous media. They contain a variety of molecular species and structural elements that may interact with each other, leading to complex lateral diffusion behavior. Insights into this behavior could help in understanding the composition, organization, and function of membranes in cell biology. Fluorescence-correlation spectroscopy (FCS) is a powerful method for determining the average diffusion coefficients of the molecules in these membranes. Measurements that are made using this approach are based on the movement of thousands of molecular transitions through an observation spot. 1 However, the diffraction-limited resolution of confocal microscopy makes it difficult to probe heterogeneity on the sub-100nm scale. Stimulated-emission-depletion (STED) microscopy has become a well-established method for achieving spatial super resolution (below 50nm). Using STED enables the size of the observation volume to be tuned. As a result, the common issue that arises from fluorescence-correlation spectroscopy, namely, the diffraction-limited lateral resolution, is avoided. This limited resolution makes it difficult to probe diffusion heterogeneity on the sub-100nm scale and is only inferable by extrapolations. By gradually shrinking the observation spot size, the type of hindered diffusion (e.g., unperturbed Brownian motion, obstacleinduced restricted diffusion, or transient trapping of molecules in permeable domains), and therefore the local environmental organization of the diffusors, can be determined.
of cryo-EM with the specificity of fluorescence. Although cryo-fluorescence microscopy suffers from optical limitations, it is a powerful way to target the resolving power of cryo-EM toward proteins of interest in heterogeneous cellular environments. Super-resolution microscopy at cryo temperatures has also been established using several different approaches. While fluorescence-derived localization is a key benefit of cryo-CLEM, fluorescence can also be used to bring orthogonal information into cryo-EM images. We recently developed a fusion assay compatible with cryo-CLEM equipment and conditions (Metskas and Briggs, Microscopy & Microanalysis 2019). This development employs auto-quenching by resonant energy transfer to specifically target a function rather than a protein in cryo-CLEM -in this case, adding information on lipid mixing to morphologies from micrographs of influenza virus fusion. However, further methods developments, particularly those involving FRET, are currently hampered by limited characterization of modern fluorophores at cryo-CLEM temperatures (77-100 K).Here, we present a study of commonly used synthetic fluorophores and fluorescent proteins, characterizing excitation and emission spectra, singlet state lifetime, and quantum yield at 77 K. We note that 10 nm shifts of the modes are common for both excitation and emission spectra, but are fluorophore specific in magnitude and even in direction. Vibronic coupling and spectral narrowing are visible in all cases characterized, and singlet state lifetimes increase or decrease in a fluorophore-specific manner. Taken together, these data suggest guidelines for choosing cryo-CLEM fluorophores and filter sets, and demonstrate promise for techniques such as FRET in carefully-adapted applications.
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