研究目的
To study the dynamics of single molecules in different fluids using a newly developed experimental scheme of fluorescence microscopy based on total internal reflection regime.
研究成果
The developed TIRFM setup successfully allowed for the study of single chlorophyll molecule dynamics in an aquatic environment. Future applications could include the study of membrane protein dynamics and their interactions with lipid bilayers, offering insights into molecular systems' properties.
研究不足
The study is limited by the diffraction limit of fluorescence microscopy, which affects the resolution of closely spaced nanoparticles. Additionally, the concentration of fluorescent molecules must be carefully controlled to ensure single-molecule detection.
1：Experimental Design and Method Selection:
The study utilized total internal reflection fluorescence microscopy (TIRFM) to investigate the dynamics of single chlorophyll molecules in an aquatic environment. The setup included a semiconductor laser, lenses, a prism for total internal reflection, an objective, an excitation filter, an EMCCD camera, and a computer for image processing.
2：Sample Selection and Data Sources:
Chlorophyll samples in low molecular concentration were selected for experimental studies to ensure single-molecule detection.
3：List of Experimental Equipment and Materials:
Semiconductor laser (wavelength 405 nm), lenses, prism, objective (NA
4：65), excitation filter, EMCCD camera (back-illuminated, pixel size 45 x 45 μm), and Altami Studio software for image display. Experimental Procedures and Operational Workflow:
The light beam from the laser was directed at the interface between two media with different refractive indices to achieve total internal reflection. Fluorophores near the surface were excited, and their fluorescence was collected by the microscope's optics. The signal was then filtered and detected by the EMCCD camera.
5：Data Analysis Methods:
ImageJ software was used for image processing, including noise elimination, segmentation, and measurement of signal intensity.
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