Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy.

Chemistry (Weinheim an der Bergstrasse, Germany) (2009-12-02)
Kirill Kolmakov, Vladimir N Belov, Jakob Bierwagen, Christian Ringemann, Veronika Müller, Christian Eggeling, Stefan W Hell
ABSTRACT

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 647N NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Sigma-Aldrich
Phalloidin–Atto 647N, BioReagent, suitable for fluorescence, ≥80% (HPLC)
Sigma-Aldrich
Anti-Rabbit-IgG - Atto 647N antibody produced in goat, 1 mg/mL IgG
Sigma-Aldrich
Atto 647N DOPE, suitable for fluorescence
Sigma-Aldrich
Anti-Mouse-IgG - Atto 647N antibody produced in goat, contains 50% glycerol as stabilizer
Sigma-Aldrich
Atto 532 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Supelco
Atto 647N, BioReagent, suitable for fluorescence, ≥98.0% (HPCE)
Supelco
Atto 647N maleimide, BioReagent, suitable for fluorescence, ≥90% (HPLC)
SAFC
Atto 465 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Supelco
NTA - Atto 647 N, BioReagent, suitable for fluorescence, ≥80% (HPCE)
Sigma-Aldrich
Atto 647 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Supelco
Atto 590 NHS ester, BioReagent, suitable for fluorescence, ≥60% (coupling to amines)
Sigma-Aldrich
Atto 647N-Streptavidin, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 425 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Sigma-Aldrich
Atto 647N Protein Labeling Kit
Sigma-Aldrich
Atto 425
Sigma-Aldrich
Atto 532, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 590-Biotin, BioReagent, suitable for fluorescence, ≥90.0% (HPCE)
Sigma-Aldrich
Phalloidin–Atto 390, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Sigma-Aldrich
Abberior® STAR 635, phalloidin, for STED application
Sigma-Aldrich
Atto 647N-Biotin, BioReagent, suitable for fluorescence, ≥90.0% (HPLC)
Sigma-Aldrich
Atto 590, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Supelco
Atto 390 NHS ester, BioReagent, suitable for fluorescence
Sigma-Aldrich
Phalloidin–Atto 532, suitable for fluorescence, ≥95.0% (HPCE)
Sigma-Aldrich
Atto 647N amine
Sigma-Aldrich
Atto 590-Streptavidin, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 647N azide
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)