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The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy
摘要： Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy F?rster resonance energy transfer (fLiM-fRet) and stimulated emission depletion (SteD) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.
关键词： FLIM-FRET,living cells,magnetotactic bacteria,STED microscopy,protein mechanics
更新于2025-09-12 10:27:22
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Background-free fluorescence decay time sensing and imaging of pH with highly photostable diazaoxotriangulenium dyes
摘要： Novel fluorescent diazaoxatriangulenium (DAOTA) pH indicators for lifetime-based self-referenced pH sensing are reported. The DAOTA dyes were decorated with phenolic receptor groups inducing fluorescence quenching via photoinduced electron transfer mechanism. Electron-withdrawing chlorine substituents ensure response in the most relevant pH range (apparent pK’a values ~5 and 7.5 for the p,p-dichlorophenol- and the p-chlorophenol-substituted dyes, respectively). The dyes feature long fluorescence lifetime (17-20 ns), high quantum yield (~60%) and high photostability. Planar optodes are prepared upon immobilization of the dyes into polyurethane hydrogel D4. Apart from the response in the fluorescence intensity, the optodes show pH-dependent lifetime behaviour which makes them suitable for studying 2D pH distribution with help of fluorescence lifetime imaging technique. The lifetime response is particularly pronounced for the sensors with high dye concentration (0.5-1% wt. in respect to the polymer) and is attributed to efficient homo-FRET mechanism.
关键词： pH sensor,photoinduced electron transfer,Decay time,triangulenium,Frequency Domain FLIM,FRET
更新于2025-09-09 09:28:46
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[Methods in Molecular Biology] BCL-2 Family Proteins Volume 1877 (Methods and Protocols) || Rapid Imaging of BCL-2 Family Interactions in Live Cells Using FLIM-FRET
摘要： The Bcl-2 proteins control cell death via interchanging interactions within the Bcl-2 family. Fluorescence lifetime imaging microscopy (FLIM) is used to detect F?rster resonance energy transfer (FRET) between two fluorescent-fusion proteins in live cells. FLIM-FRET has been used to detect specific interactions and their disruption, for Bcl-2 family proteins. To date, this has been possible only in low throughput, due to the time required for serial data acquisition. We developed an automated optical system with eight parallel detectors for rapid and efficient data collection. Here we describe how to use this system for FLIM-FRET imaging of Bcl-2 family protein interactions in a 384-well plate format.
关键词： BH3 mimetic,FLIM Hyperspectral,FLIM-FRET,Bcl-2 family,mCerulean3,High throughput,Fluorescence lifetime imaging microscopy
更新于2025-09-04 15:30:14