have shown that GFP can be rapidly and efficiently photoconverted in plant, Drosophila, and mammalian cells. found that only the protonated form of GFP is photoconverted, so this technique may not be appropriate for high-pH cellular compartments. For low-abundance GFP fusions, a photoconversion signal may not be observed. Red signal intensity increases with GFP concentration and saturates around 30 uM. One concern with photoconverting GFP is the expression level necessary to observe a signal. 2015 CC BY 4.0 Applications and limitations of GFP photoconversion Photoconverted GFP spreads through the cytosol via cytoplasmic strands. White circles represent the irradiated area. Tobacco suspension cell culture with cytosol-targeted GFP. This imaging process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red color was observed after excitation at 488 nm, indicating that spontaneous conversion did not occur. ![]() They subsequently confirmed that purified GFP could be imaged in the green state after excitation at 488 nm, photoconverted with 405 nm light, and then captured in the red state after excitation at 561 nm. After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. Like many great scientific discoveries, this one was made by accident. ![]() ’s discovery that GFP itself is photoconvertible, new studies of spatiotemporal dynamics can be undertaken using available GFP lines. In many cases, however, a GFP-tagged cell line or organism has already been made, and it would be expensive and time-consuming to repeat the process with these new photoconvertible proteins. ![]() Newly developed photoconvertible FPs like PsMOrange and mEos add another layer of complexity to imaging, as they allow one to monitor compartment/protein dynamics or evaluate protein turnover rate. Even though GFP has been so thoroughly characterized, it turns out this protein has a few more secrets - during a collaboration, members of Maureen Hanson’s and Rima Menassa's labs made the accidental discovery that laser treatment can photoconvert GFP from green to RED! This simple technique has been shown to work in plant, Drosophila and mammalian cells, and it may find wide use in biological research. Fluorescent proteins (FPs) are also the foundation of multiple important technologies, including FRET and optogenetics. Chances are you’ve used a GFP derivative to conduct subcellular localization studies or make a reporter construct. Since the first research applications of GFP were published in the 1990s, biologists have spent a lot of time making things glow.
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