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Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging.

Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology (2009-04-02)
Sebastian van de Linde, Ulrike Endesfelder, Anindita Mukherjee, Mark Schüttpelz, Gerd Wiebusch, Steve Wolter, Mike Heilemann, Markus Sauer
ABSTRACT

We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with approximately 20 nm optical resolution.

MATERIALS
Product Number
Brand
Product Description

Supelco
Atto 565 NHS ester, BioReagent, suitable for fluorescence, ≥70.0% (coupling to amines)
Sigma-Aldrich
Atto 565, BioReagent, suitable for fluorescence, ≥75% (sum of isomers, HPCE/HPLC)
Sigma-Aldrich
Atto 565-Biotin, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 655, BioReagent, suitable for fluorescence, ≥85% (HPLC)
Sigma-Aldrich
Atto 655 NHS ester, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 655-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Sigma-Aldrich
Atto 565 maleimide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 700 NHS-ester, BioReagent, suitable for fluorescence, ≥90% (coupling to amines)
Sigma-Aldrich
Atto 655 amine, suitable for fluorescence
Sigma-Aldrich
Atto 700 azide, suitable for fluorescence
Sigma-Aldrich
Atto 565 azide, BioReagent, suitable for fluorescence, ≥80.0% (HPCE)
Sigma-Aldrich
Atto 565 iodoacetamide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Anti-Cytochrome c antibody produced in sheep, ~0.5 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Atto 700 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 700, BioReagent, suitable for fluorescence, ≥96.0% (HPCE)
Sigma-Aldrich
Atto 655-maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling rate)

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