研究目的
To propose and demonstrate a super-resolution imaging scheme in the fluorescence lifetime domain using lifetime blinking and high-order cumulant analysis.
研究成果
ltSOFI enables super-resolution imaging in the fluorescence lifetime domain by analyzing lifetime fluctuations, achieving spatial resolution below 100 nm in simulations and 176 nm in experiments. It offers a new dimension for super-resolution microscopy without hardware modifications.
研究不足
The study is based on numerical simulations and a tentative experimental demonstration; further validation with more diverse samples and real-time applications may be needed. The method relies on specific fluorophores with lifetime blinking properties.
1：Experimental Design and Method Selection:
The study uses numerical simulations and experimental data analysis to validate the ltSOFI method, which leverages fluorescence lifetime blinking and high-order cumulant analysis for super-resolution imaging.
2：Sample Selection and Data Sources:
Numerical simulations involve test objects with emitters exhibiting lifetime fluctuations; experimental data comes from time-lapse FLIM recordings of HeLa cells expressing a cAMP sensor.
3：List of Experimental Equipment and Materials:
Includes a gated optical intensifier for FLIM, CCD camera for intensity measurement, and software like Localizer in Igor Pro for reconstruction.
4：Experimental Procedures and Operational Workflow:
Involves pulsed excitation and gated detection for lifetime measurement, recording image sequences, applying exponential fitting for lifetime calculation, and performing cumulant analysis on multiplied intensity and lifetime fluctuation images.
5：Data Analysis Methods:
Uses high-order cumulant analysis, Fourier Ring Correlation (FRC) for resolution evaluation, and software tools such as NanoJ-SQUIRREL in ImageJ.
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