Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Summary

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

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.

Protocol

1. Preparation of fluorescent protein droplet samples

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. (Optionally, after cleaning the glassware can be treated for 30 seconds in a plasma cleaner to obtai....

Discussion

The purified fluorescent protein droplet sample is a very convenient sample format for the photophysical characterization of fluorescent proteins, for example to study photobleaching kinetics and photoconversion kinetics. The extremely small droplet volume (~20 picoliter) facilitates photobleaching and photoconversion experiments, which can be difficult to perform in cuvette based systems. In addition, as shown here the droplet sample is ideally suited for setting up a confocal microscope for photoconversion applicatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microsope slides	VWR international	48312-003
22 mm cover glass	Corning	2940-245
1-octanol	Sigma-Aldrich	O4500
methyltrimethoxysilane	Sigma-Aldrich	M6420
MatTek dishes	MatTek Corp.	P35G-1.5-14-C
Lipofectamine2000	Invitrogen	11668-019