研究目的
To develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution.
研究成果
The FLIM-DSLM system provides a convenient and broadly applicable platform for relatively large sample imaging and facilitates the 3D mapping and quantitative analysis of functional information for biomedical and other research fields. It can be utilized to monitor protein-protein interactions via FRET on different spatial scales.
研究不足
The current system requires multiple acquisitions and post-processing using the stitching method for larger samples, presenting challenges in terms of data management and image processing. The pixel size and magnification can be changed according to the imaging requirements, but this affects the field of view and spatial resolution.
1：Experimental Design and Method Selection:
The FLIM-DSLM system was designed to acquire a series of time-gated fluorescence intensity images at different time delay positions with respect to the excitation pulses at different Z positions. The lifetime was determined for each voxel by iteratively fitting to single exponential decay.
2：Sample Selection and Data Sources:
A lifetime reference Rhodamine 6G solution and a sub-resolution fluorescent bead phantom were used to evaluate the system's performance. Tg(kdrl:EGFP) transgenic zebrafish embryos were imaged ex vivo and in vivo.
3：List of Experimental Equipment and Materials:
A spectrally filtered ultrafast fiber-laser-pumped super-continuum source, a digital scanning system, a gated optical intensifier (GOI), and a sCMOS camera were used.
4：Experimental Procedures and Operational Workflow:
The GOI was gated with an adjustable delay relative to the excitation pulses, and the resulting time-gated and intensified fluorescence signals were recorded by the sCMOS camera.
5：Data Analysis Methods:
The 3-D lifetime distribution was determined by assuming a single exponential fluorescence decay model with background subtraction and IRF deconvolution using the FLIMfit software tool.
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