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.
Highlights
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light
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 Photoconvertible fluorescent proteins (pc-FPs) photoconversion applications
A fluorescent protein droplet sample consists of a 1-octanol/water emulsion with the fluorescent protein residing in the water phase
Summary
A fluorescent protein droplet sample consists of a 1-octanol/water emulsion with the fluorescent protein residing in the water phase. This emulsion is sandwiched between a microscope slide and a 22 mm square cover glass for microscopy applications. 1. Before making fluorescent protein droplet samples the microscope slides and cover glasses need to be cleaned and coated with a hydrophobic agent. 2. Clean glassware by washing 5 minutes with acetone and leave to dry by air. 5. To make the emulsion pipette 45 μl 1-octanol and 5 μl fluorescent protein in an microfuge tube. After sonication pipette 4 μl emulsion from the middle of the tube onto a coated microscope slide and cover with a coated cover glass. The largest droplets are close to the center of the sample and the smaller are located further towards the edges
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