研究目的
Investigating how laser beams affect fluorescence lifetime in both confocal and STED imaging modes to improve STED imaging resolution.
研究成果
The study demonstrated that increasing fluorescence lifetime by prolonging illumination time in STED imaging mode can improve resolution at the same depletion power. This phenomenon was observed in fluorescent microspheres and biological samples, suggesting potential for synthesizing fluorescent labels sensitive to environmental changes for enhanced STED nanoscopy resolution.
研究不足
The increased fluorescence lifetime leads to resolution improvement but also causes photobleaching, reducing signal intensity. The balance between resolution and signal intensity is a limitation.
1：Experimental Design and Method Selection:
A STED-FLIM imaging system was developed to simultaneously collect intensity and lifetime signals of fluorescent labels. The system included excitation and depletion beams, with the fluorescence lifetime changes observed in confocal and STED imaging modes.
2：Sample Selection and Data Sources:
Two kinds of fluorescent microspheres (40 nm and 100 nm in diameter) and Star635P labeled human Nup153 in fixed HeLa cells were used.
3：List of Experimental Equipment and Materials:
The system included a picosecond diode laser, an ultrafast Ti:sapphire laser, a photomultiplier tube, and various optical components like lenses, mirrors, and fibers.
4：Experimental Procedures and Operational Workflow:
The samples were imaged in confocal and STED modes, with fluorescence lifetime measured over increasing illumination times.
5：Data Analysis Methods:
Fluorescence lifetime was analyzed using SPCM software, with resolution improvement evaluated through full-width at half-maximum (FWHM) measurements.
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