研究目的
To develop a new FRET pair that allows concomitant STED imaging of one of its constituents in living cells and to study the mechanics of the MamJ/MamK pair in magnetotactic bacteria.
研究成果
The study successfully developed a new FRET pair for correlative FLIM-FRET and STED microscopy in living cells, revealing that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. The technique is anticipated to have wide applicability in studying biological processes in living cells at high resolution.
研究不足
The study is limited by the photodamage and phototoxicity associated with STED microscopy in living cells, and the potential influence of fluorescent protein tags on the mechanics of the studied proteins.
1:Experimental Design and Method Selection:
The study combines FLIM-FRET and STED microscopy to study protein mechanics and structure in living cells.
2:Sample Selection and Data Sources:
Magnetospirillum gryphiswaldense MSR-1 bacteria were used as a model system.
3:List of Experimental Equipment and Materials:
Includes a MicroTime 200 time-resolved confocal fluorescence microscope, a ps-pulsed supercontinuum source, and a single-photon avalanche diode.
4:Experimental Procedures and Operational Workflow:
Bacteria were grown, harvested, and suspended in a buffer. FLIM-FRET and STED microscopy were performed before and after applying a magnetic torque.
5:Data Analysis Methods:
Data were analyzed using deconvolution fits, spectral overlap integrals, and F?rster distance calculations.
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