Protocols used in Molecular Biology

Dictyostelium Discoideum: Live Cell Imaging in Changing Perspective

Author(s): Abhishek Singh

Pp: 126-145 (20)

DOI: 10.2174/9789811439315120010016

* (Excluding Mailing and Handling)


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.

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