Department of Molecular Physiology and Biophysics, Vanderbilt University
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Kremers, G., Piston, D. Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells. J. Vis. Exp. (40), e1995, doi:10.3791/1995 (2010).
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.
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.
2. Setting up a photoconversion experiment
The following procedure is a general strategy for setting up a fluorescent protein photoconversion experiment. This procedure can be applied for purified proteins as well as for live cells.
3. Dual-probe optical highlighting with mOrange2 and Dronpa
Because of the red-shifted spectral properties, mOrange2 can be used in combination with the green photoswitchable fluorescent protein Dronpa for dual-probe optical highlighting to allow selective highlighting of 4 individual cell(organelle) populations.
4. Representative Results
Figure 1. Droplet sample preparation. A) Correctly prepared droplet sample. B) Sample prepared without coating the microscope slide and cover glass. C) Sample prepared without adding 0.1% BSA.
Figure 2. Effect of photoconversion laser power and duration on mOrange2 photoconversion. Single droplets containing mOrange2 protein were continuously photoconverted using different amounts of 488 nm laser power. Relative laser power used for photoconversion was 10% (solid), 25% (dashed), and 100% (dotted). A) Orange fluorescent species. B) Photoconverted red fluorescent species.
Figure 3. Dual-probe optical highlighting with mOrange2 and Dronpa. A) Cell expressing mOrange2-Histone H2B and Dronpa-Mito before photoconversion, showing orange fluorescence in the nucleus and green fluorescence in the mitochondria. B) Dronpa fluorescence was switched off with low power 488 nm excitation, causing minimal photoconversion of mOrange2. C) mOrange2 was photoconverted to red with high power 488 nm excitation. D) Dronpa fluorescence was switched on again using 800 nm 2-photon excitation. The panels are overlays of the fluorescence images together with the differential interference contrast image.
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 applications. The hydrophobic coating and the presence of BSA are important for obtaining homogeneous droplets. Without coating the droplets tend to be squashed against one of the glass surfaces and in the absence of BSA the fluorescent protein tend to accumulate at the 1-octanol/water interface, creating a halo of fluorescence (Figure 1).
Fluorescent protein photoconversion is often regarded as an alternative to fluorescence recovery after photobleaching (FRAP), with the advantage that one can also follow the photoconverted species. However, it is important to consider that photoconversion is critically dependent on the laser power used. Too much photoconversion laser power or too long exposure will cause photobleaching rather than photoconversion, thereby reducing the amount of photoconverted fluorescence (Figure 2).
The red-shifted spectral properties of mOrange2 and its photoconverted species permit dual-probe optical highlighting with a green fluorescent optical highlighter. This can either be a photoswitchable fluorescent protein (Dronpa), as demonstrated here, or alternatively a photoactivatable fluorescent protein, for example PA-GFP. Dronpa has the advantage that one can check its presence at the start of the experiment. On the other hand, the use of Dronpa complicates optical highlighting, because all Dronpa fluorescence has to be inactivated first, and the fact that Dronpa fluorescence is gradually switched off during imaging. These complications are less profound when using PA-GFP, but checking for the presence of PA-GFP before photoactivation can be more difficult.
No conflicts of interest declared.
We thank Mike W. Davidson (Florida State University) for providing plasmid DNA encoding fluorescent proteins. This work was supported by National Institutes of Health grant GM72048 (to D.W.P.).
|Microsope slides||VWR international||48312-003|
|22 mm cover glass||Corning||2940-245|
|MatTek dishes||MatTek Corp.||P35G-1.5-14-C|