The advent of advanced microscopes; during microscope evolution from
simple microscopes to confocal and live cell microscope; having digital imaging
facility revolutionized our view for the living cells. In the protein localization study,
fluorescent proteins are tagged at amino or carboxyl (preferably) terminal of desired
protein for live cell study. These live cell studies improved our understanding of
protein dynamics and understanding its role in biological regulation. The mutational
variants of fluorescent tags (GFP, RFP); can be used with different protein; which will
efficiently use UV-Visible to Far Red light spectrum; without overlapping of excitation
and emission spectrum. Further, various cell organelle (Lysosome, Golgi bodies,
Endoplasmic Reticulum, Mitochondria, Nucleus) trackers; improved our live cell
localization studies in the wide non-overlapping UV-Visible spectrum.This chapter
gives an overview for live cell protein localization study in mitotically active,
unicellular stage of Dictyostelium discoideum. This evolutionary cutting edge organism
had both unicellular as well as multicellular stages during its life cycle. This chapter
will provide the design of fusion of fluorescent tag to the specific gene and its live cell
localization. Further, it will cover; transformation of the unicellular organism; drug
based selection; sample preparation with nuclear, mitochondrial localization markers
(trackers) and live cell localization study on live cell-confocal microscope setup. It will
also have a glimpse of the design of fusion protein with an aspect of advantage and
disadvantages.
Keywords: Confocal, DAPI, Dictyostelium discoideum, EMCCD, Fluorescent
proteins GFP, Golgo bodies, RFP, Live cell imaging, MitoTracker, Mitochondria,
UV-visible spectrum.
