Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

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.

Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells

“…1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Ganglioside GM1 (Ovine Brain) were purchased from Avanti (Avanti Polar Lipids Inc., Alabaster, Alabama, USA). Cholera Toxin B (CtxB) subunit was purchased from Sigma-Aldrich and labelled with the amine reactive dye KK114-NHS 24 (now AbberiorSTAR, Abberior) according to the standard protocol, resulting in less than or equal to two dyes per CtxB. The two lipid analogues labelled with the organic dye Atto647N, N-(Atto647N)-1,2-dipalmitoyl-sn-glycero-3-PE (head-group labelling; DPPE-Atto647N or simply PE) and N-(Atto647N)-sphingosylphosphocholine (acyl-chain replacement; SM-Atto647N or simply SM) were purchased from Atto-Tec (ATTO-TEC GmbH, Siegen, Germany).…”

“…The two lipid analogues labelled with the organic dye Atto647N, N-(Atto647N)-1,2-dipalmitoyl-sn-glycero-3-PE (head-group labelling; DPPE-Atto647N or simply PE) and N-(Atto647N)-sphingosylphosphocholine (acyl-chain replacement; SM-Atto647N or simply SM) were purchased from Atto-Tec (ATTO-TEC GmbH, Siegen, Germany). The lipids labelled with the organic dye KK114, PE-KK114, DSPE-PEG(2k)-KK114 (or simply DSPE-PEG, head-group labelling), DOPE-PEG(2k)-KK114 (or simply DOPE-PEG, head-group labelling) and Cholesterol-PEG(1k)-KK114 (or simply Chol-PEG) were synthesized as described previously 23 using starting compounds DSPE-PEG(2000)-NH2, DOPE-PEG(2000)-NH2 (Avanti Polar Lipids), Cholesterol-PEG(1000)-NH2 (Nanocs Inc., NY, USA) and the amine reactive dye KK114-NHS 24 . The molecular structures of all fluorescent lipid analogues are depicted in Fig.…”

Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA

“…for 5 min at −20°C (for DNA labeling), extracted with 0.5% (vol/vol) Triton X-100 in PBS, blocked with 5% (wt/vol) BSA in PBS, and incubated with monoclonal mouse antibodies against TFAM (Abnova), DNA (PROGEN), or TOM20 (Santa Cruz). The primary antibodies were detected with secondary antibodies (sheep anti-mouse and goat anti-rabbit; Jackson Immuno Research Laboratories) custom-labeled with ATTO532 (AttoTec) or KK114 (36). After immunolabeling, the samples were mounted in MOWIOL with 0.1% (wt/vol) 1,4-Diazabicyclo [2.2.2]octane (DABCO) and 2.5 μg/mL DAPI (Sigma Aldrich).…”

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca V 1.3 Ca 2+ channels at hair cell active zones

“…Two-color STED images were acquired on a custom-built setup, as described previously (57). A pair of pulsed laserdiodes at 595 and 640 nm wavelength were used to excite the dyes Atto590 (Atto-Tec) and KK114 (58).…”