PAmCherry and converted mEos3.2), severely implicating multicolor super‐resolution imaging. In addition, mEos3.2 demonstrates excellent performance in PALM imaging in terms of its brightness, maturation time and labelling density 13. mEos3.2 is truly monomeric, which is crucial for BiFC. Importantly, the blue‐shifted derivatives emit in the spectral range of the red fluorescent proteins (e. g. Instead, we chose mEos3.2, a recently developed photoconvertible fluorescent protein 13, as the BiFC-PALM probe. The dynamics of this reaction are altered by the presence of reducing agents. In the case of Alexa Fluor 647, the most commonly used fluorescent label in super‐resolution microscopy, this derivative is created over time in an intramolecular, irreversible photoinduced chemical reaction. Herein we show that upon intense irradiation with a 561 nm laser line, far‐red organic dyes photoconvert to blue‐shifted emissive species. However, irradiation with high laser intensity can induce photo‐conversion of some of the most frequently used fluorophores. Combining Primed Photoconversion and UV-Photoactivation for. A far‐red emitting dye is often one of the labels of choice. In a multicolor approach, adequate spectral separation of the different photoswitchable probes is required. Localization‐based super‐resolution microscopy has become an indispensable tool in biology to study features smaller than the diffraction limit of light. mEos3.2 and PA-JF 646 were photoconverted by 100 long excitation pulses of 407.
Photoconversion of Far‐Red Organic Dyes: Implications for Multicolor Super‐Resolution Imaging Lieve Dirix, Koen Kennes, Eduard Fron, Zeger Debyser, Mark Van der Auweraer, Johan Hofkens, Susana Rocha (see publication in Journal ) Abstract The median number of detected photons per mEos3.2 molecule per frame was.
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