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Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses.

Optics express (2011-03-04)
Johanna Bückers, Dominik Wildanger, Giuseppe Vicidomini, Lars Kastrup, Stefan W Hell
ABSTRACT

We describe a STED microscope optimized for colocalization experiments with up to three colors. Two fluorescence labels are separated by their fluorescence lifetime whereas a third channel is discriminated by the wavelength of fluorescence emission. Since it does not require a second STED beam, separating by lifetime is insensitive to drift and thus optimally suited for colocalization analyses. Furthermore, we propose a setup having a second STED beam for long duration multicolor recording.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 647N NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
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SAFC
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Atto 647 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Supelco
NTA - Atto 647 N, BioReagent, suitable for fluorescence, ≥80% (HPCE)
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Atto 647N-Streptavidin, BioReagent, suitable for fluorescence
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Atto 590 NHS ester, BioReagent, suitable for fluorescence, ≥60% (coupling to amines)
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Atto 425 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
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Atto 647N Protein Labeling Kit
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Atto 532, BioReagent, suitable for fluorescence, ≥90% (HPCE)
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Atto 590-Biotin, BioReagent, suitable for fluorescence, ≥90.0% (HPCE)
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Atto 647N-Biotin, BioReagent, suitable for fluorescence, ≥90.0% (HPLC)
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Atto 590, BioReagent, suitable for fluorescence, ≥90% (HPCE)
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Phalloidin–Atto 390, BioReagent, suitable for fluorescence, ≥90% (HPLC)
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Atto 390 NHS ester, BioReagent, suitable for fluorescence
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Abberior® STAR 635, phalloidin, for STED application
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Phalloidin–Atto 532, suitable for fluorescence, ≥95.0% (HPCE)
Sigma-Aldrich
Atto 590-Streptavidin, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 647N amine
Sigma-Aldrich
Phalloidin–Atto 425, suitable for fluorescence, ≥90% (HPLC)
Sigma-Aldrich
Atto 647N azide
Sigma-Aldrich
Phalloidin–Atto 590, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 425 maleimide, BioReagent, suitable for fluorescence, ≥70% (coupling to thiols)
Sigma-Aldrich
Atto 425-Streptavidin, BioReagent, suitable for fluorescence, ≥95% (HPCE)
Sigma-Aldrich
Atto 390 azide, BioReagent, suitable for fluorescence, ≥90% (HPLC)