Kirill Kolmakov

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

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.

Novel red fluorophores with superior performance in STED microscopy

In optical microscopy, most red-emitting dyes provide only moderate performance due to unspecific binding, poor labeling efficiency, and insufficient brightness. Here we report on four novel red fluororescent dyes, including the first phosphorylated dye, created by combining a rigidized rhodamine backbone with various polar groups. They exhibit large fluorescence quantum yields and improved NHS ester stability. While these fluorophores are highly suitable for fluorescence microscopy in general, they excel in stimulated emission depletion (STED) microscopy, providing < 25 nm spatial resolution in raw images of cells.

Polar Red-Emitting Rhodamine Dyes with Reactive Groups: Synthesis, Photophysical Properties, and Two-Color STED Nanoscopy Applications†

The synthesis, reactivity, and photophysical properties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the amino-reactive sites (NHS esters or mixed N-succinimidyl carbonates) are reported. All fluorophores contain an N-alkyl-1,2-dihydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary carboxamide group. The absorption and emission maxima in water are in the range of 635-639 and 655-659 nm, respectively. A vastly simplified approach to red-emitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed. As an example, a phosphorylated dye with an azide residue was prepared and was used in a click reaction with a strained alkyne bearing an N-hydroxysuccinimid (NHS) ester group. This method bypasses the undesired activation of phosphate groups, and gives an amphiphilic amino-reactive dye, the solubility and distribution of which between aqueous and organic phases can be controlled by varying the pH. The presence of two hydroxyl groups and a phenyl ring with two carboxyl residues in the dyes with another substitution pattern is sufficient for providing the hydrophilic properties. Selective formation of a mono-N-hydroxysuccinimidyl ester from 5-carboxy isomer of this rhodamine is reported. The fluorescence quantum yields varied from 58 to 92% for free fluorophores, and amounted to 18-64% for antibody conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two-color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting a pair of fluorophores with absorption/emission bands at 579/609 and 635/655 nm enabled two-color channels with low cross-talk and negligible background at approximately 40 nm resolution.

A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

Far-red emitting fluorescent dyes for optical nanoscopy: Fluorinated silicon–rhodamines (SiRF dyes) and phosphorylated oxazines.

Far-red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground-state depletion (GSDIM) super-resolution microscopy are presented. Fluorinated silicon-rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high-yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon-rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (<600 Da). The use of the λ=800 nm STED beam instead of the commonly used one at λ=750-775 nm provides excellent imaging performance and suppresses re-excitation of SiRF and the oxazine dyes. The photophysical properties and immunofluorescence imaging performance of these new far-red emitting dyes (photobleaching, optical resolution, and switch-off behavior) are discussed in detail and compared with those of some well-established fluorophores with similar spectral properties.

Hydroxylated fluorescent dyes for live-cell labeling: Synthesis, spectra and super-resolution STED.

Abstract Hydroxylated rhodamines, carbopyronines, silico‐ and germanorhodamines with absorption maxima in the range of 530–640 nm were prepared and applied in specific labeling of living cells. The direct and high‐yielding entry to germa‐ and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila‐ and germafluoresceins, as well as ‐rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50–75 nm in one‐ and two‐color imaging of vimentin‐HaloTag fused protein and native tubulin. The established structure–property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo‐, silico‐, and germanorhodamines using simple additive schemes.

Author Correction: A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization.

This corrects the article DOI: 10.1038/ncomms10144.

Corrigendum: A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

This corrects the article DOI: 10.1038/ncomms10144.