Scanning FCS (sFCS) is a great tool for studying slowly diffusing species as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly through the sample allowing for simultaneous measurement at multiple locations. The shorter residence times also lead to lower photon doses experienced by each detected molecule, reducing the risk of photobleaching. Here, we show results from sFCS measurements on supported lipid bilayers (SLBs) where fluorescence lifetime information is used to achieve an axial nanometric localization based on Metal Induced Energy Transfer (MIET).
Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential for imaging dynamic processes in life science. The rapidFLIM approach dramatically reduces acquisition times through a combination of fast beam scanning, hybrid photomultiplier detectors, which are capable of handling very high count rates, and TCSPC modules with ultra short dead times. With this hardware combination, excellent photon statistics can be achieved in significantly shorter time spans, allowing fast processes to be measured with the high spatial resolution offered in confocal microscopy. Depending on image size, rapidFLIM enables following dynamic processes like protein interactions, chemical reactions or highly mobile species in live cell imaging with a rate of several frames per second.
The separation of overlapping fluorescence emissions in biological samples has been improved in the last years by using spectral confocal microscopy in combination with linear unmixing. However, the separation of multiple labels in biological samples remains challenging, especially when strong tissue autofluorescence (AF) overshadows specifically labeled structures. Combining the spectral approach with fluorescence lifetime measurements based on a simultaneous acquisition of both spectral and lifetime parameters could significantly improve the separation quality between multiple labels and tissue AF. We demonstrate this approach in highly autofluorescent human lung tissue, where the fluorescence signals from specific stainings are sometimes weaker than tissue AF. We use dual color Pulsed Interlevaed Excitation (PIE) in conjunction with a spectral FLIM (sFLIM) detection system featuring eight separate TCSPC timing channels and analyze the data by applying a unique pattern matching technique.
Fluorescence Correlation Spectroscopy (FCS) is an important technique for understanding molecular dynamics and motion on timescales ranging from nanoseconds to seconds. The high concentrations found in some biological systems reduce the significance of FCS, as too many fluorophores are present within a standard confocal volume. Combining FCS with Stimulated Emission Depletion (STED) is one technique to overcome this problem by reducing the observation volume in the sample, reducing the number of molecules within this volume. This technique also allows the observation volume to be tuned to access additional information, such as the parameters which characterize hindered diffusion.
In addition, by utilizing galvoscanners, scanning FCS can increase the number of transits recorded and reduce the residence time of each molecule, increasing the statistics and reducing the effects of photobleaching respectively. This technique is also useful for increasing the number of transits observed for slowly diffusing species at low concentrations, such as observed in membranes.
Measuring multiple species simultaneously saves time and allows interactions between molecules to be investigated, which may not be clear from multiple experiments investigating single species. By exploiting the lifetime information available from a microscope equipped with Time Correlated Single Photon Counting (TCSPC) hardware, pattern matching can be used to separate similar fluorophores allowing up to three superresolved species to be resolved using a single STED laser. This pattern matching analysis can be combined with STED-FCS and scanning FCS to investigate complex diffusion in membranes.
Using time-correlated single photon counting 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 artifacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remain an issue to be addressed. We propose a data analysis method correcting for this type of artifact 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 shows a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescence lifetime imaging 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.
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