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Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells.
J Vis Exp. 2010 Jun 26JV

Abstract

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles. Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion. This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

Authors+Show Affiliations

Department of Molecular Physiology and Biophysics, Vanderbilt University, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Video-Audio Media

Language

eng

PubMed ID

20613710

Citation

Kremers, Gert-Jan, and David Piston. "Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells." Journal of Visualized Experiments : JoVE, 2010.
Kremers GJ, Piston D. Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells. J Vis Exp. 2010.
Kremers, G. J., & Piston, D. (2010). Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells. Journal of Visualized Experiments : JoVE, (40). https://doi.org/10.3791/1995
Kremers GJ, Piston D. Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells. J Vis Exp. 2010 Jun 26;(40) PubMed PMID: 20613710.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells. AU - Kremers,Gert-Jan, AU - Piston,David, Y1 - 2010/06/26/ PY - 2010/7/9/entrez PY - 2010/7/9/pubmed PY - 2010/8/10/medline JF - Journal of visualized experiments : JoVE JO - J Vis Exp IS - 40 N2 - Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles. Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion. This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa. SN - 1940-087X UR - https://www.unboundmedicine.com/medline/citation/20613710/Photoconversion_of_purified_fluorescent_proteins_and_dual_probe_optical_highlighting_in_live_cells_ L2 - https://doi.org/10.3791/1995 DB - PRIME DP - Unbound Medicine ER -