Which microscopy is best to track protein movement in a living cell using GFP?
Green fluorescent protein fused to a protein of interest allows non-invasive tracking inside living cells, but requires a system that rejects background and enables time-lapse. Confocal microscopy uses laser excitation with pinhole filtering to capture sharp optical sections through living cytoplasm, minimizing photodamage compared to widefield while providing high signal-to-noise. Time-lapse acquisition of GFP dynamics reveals trafficking between endoplasmic reticulum, Golgi and plasma membrane, cytoskeletal assembly and nuclear import. Alternative modalities like bright field lack molecular
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.