Primed Conversion: The New Kid on the Block for Photoconversion

Abstract

In 2015, a novel way to convert photoconvertible fluorescent proteins was reported that uses the intercept of blue and far-red light instead of traditional violet or near-UV light illumination. This Minireview describes and contrasts this distinct two-step mechanism termed primed conversion with traditional photoconversion. We provide a comprehensive overview of what is known to date about primed conversion and focus on the molecular requirements for it to take place. We provide examples of its application to axially confined photoconversion in complex tissues as well as super-resolution microscopy. Further, we describe why and when it is useful, including its advantages and disadvantages, and give an insight into potential future development in the field.

Between life and death: strategies to reduce phototoxicity in super-resolution microscopy

“…Primed conversion is another promising UV-independent approach to induce photoswitching (figure 4(a) , right) [ 71 ]. Thereby a combination of blue and near-infrared illumination induces photoconversion in Dendra2 and the newly developed primed-conversion protein pr-mEos2 [ 71 , 72 ]. Recently, a general mechanism for primed conversion was described, which is anticipated to accelerate the development of more FPs that can be photoconverted with this live-cell friendly approach [ 73 ].…”

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

“…4a, right). Thereby a combination of blue and near-infrared illumination induces photoconversion in Dendra2 and the newly developed primedconversion protein pr-mEos2 (71,72). Recently, a general mechanism for primed conversion was described, which is anticipated to accelerate the development of more FPs that can be photoconverted with this live-cell friendly approach (73).…”

Characterization of mApple as a Red Fluorescent Protein for Cryogenic Single-Molecule Imaging with Turn-Off and Turn-On Active Control Mechanisms

“…Switching in response to simultaneous illumination has been reported in other FPs previously at RT and is known as primed conversion . This is not unlike previously reported two-color mechanisms of low-temperature photochemistry. , In primed conversion, the reported mechanism is that the readout laser produces a population of emitters in a triplet state that can then be excited again with another wavelength to produce photoswitching. , It is unclear in our case if the intermediate state is a triplet or some other long-lived intermediate stabilized at cryogenic temperatures and detailed assignment of that state is beyond the scope of this work. In this work, we show that mApple can be moved into a dark state with sufficient efficiency to create the required low active emitter concentrations for single-molecule imaging, as well as stochastic and photoactivatable turn-on mechanisms allowing for the subsequent localization of individual emitters.…”