研究目的
To introduce and evaluate PIE-based line-scanning spatial correlation spectroscopy (PIE-lsSCS) as a quantitative fluorescence microscopy method for studying dynamics in free-standing lipid bilayer membranes.
研究成果
PIE-lsSCS is a reliable and versatile technique for quantifying diffusion in free-standing 2D membranes, offering advantages such as reduced photobleaching and more efficient sampling over larger regions. It is particularly beneficial for studies involving live cells and tissues.
研究不足
The spatial resolution of PIE-lsSCS is lower than that of single-spot lsFCS, which may not be suitable for studies requiring high spatial resolution. The method's effectiveness is dependent on careful adjustment of sampling parameters to match the dynamics measured.
1：Experimental Design and Method Selection
The study employs a confocal microscope to perform repetitive, perpendicular line scans through a free-standing vertical membrane. The method utilizes pulsed interleaved excitation (PIE) for dual-color excitation within a single line scan, avoiding channel crosstalk. Spatial correlation analysis is used to quantify the diffusion of fluorescent molecules within the membrane.
2：Sample Selection and Data Sources
Experiments were conducted on fluorescently labeled giant unilamellar vesicles (GUVs) and membrane-stained live cells (NCI-H1703).
3：List of Experimental Equipment and Materials
Confocal microscope (DMi8, Leica Microsystems), picosecond pulsed lasers (470 nm, 561 nm, 640 nm), avalanche photodiodes (APD1 and APD2), TCSPC card (SPC-150), fluorescent dyes (Atto488, Atto565, Atto647N-DPPE, DiI), CellMask Green and CellMask Deep Red plasma membrane stains.
4：Experimental Procedures and Operational Workflow
The membrane is scanned multiple times along the x-axis perpendicular to the membrane, each time slightly displaced along the y-axis. Fluorescence is excited by two spatially overlapped, focused laser beams. The arrival time of each detected photon is recorded for unambiguous assignment to the correct color channel.
5：Data Analysis Methods
Spatial correlation analysis is performed on the integrated membrane intensities along the y-axis. The analysis includes background subtraction, removal of immobile structures, and normalization of each xy-frame.
独家科研数据包，助您复现前沿成果，加速创新突破
获取完整内容
