研究目的
Investigating the diffusive properties of single molecules of the photo-switchable fluorescent protein Dreiklang (DRK) inside the cell nucleus of Chinese hamster ovary (CHO) cells using laser switching contrast microscopy (LSCM).
研究成果
LSCM is a powerful technique for investigating the mobility of molecules in biological structures, particularly when conducted with the switchable fluorescent protein DRK. The study supports the model of the cell interior as a random obstacle model with areas of free diffusion and areas that restrict diffusion. The technique offers potential for further exploration of nanofluidic phenomena in living material.
研究不足
The study is limited by the assumptions made in the diffusion models, such as constant diffusion coefficients and the neglect of potential rotation of the fluid. The complexity of the cell cytosol may also introduce uncertainties in the interpretation of the results.
1：Experimental Design and Method Selection:
The study employs LSCM to monitor the dynamics of DRK molecules in CHO cells. The technique involves photo-switching DRK into a dark state with a focused laser beam and observing the recovery of fluorescence.
2：Sample Selection and Data Sources:
CHO cells expressing DRK in the cytosol were used. The cells were transiently transfected with DRK cDNA.
3：List of Experimental Equipment and Materials:
A Nikon TI Eclipse microscope equipped with different light sources, excitation laser wavelengths, and detectors was used. The setup included pulsed diode lasers for excitation and an EM-CCD based wide field camera for detection.
4：Experimental Procedures and Operational Workflow:
The DRK molecules were photo-switched into a dark state with a focused 405 nm laser beam, and the recovery of fluorescence was monitored over time.
5：Data Analysis Methods:
The diffusive behavior of DRK molecules was analyzed by fitting the recovery trajectories of fluorescence intensity to models of free and restricted diffusion.
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