Vladimir N. Belov

Direct observation of the nanoscale dynamics of membrane lipids in a living cell

Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains (‘rafts’) remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5–200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes ∼70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently (∼10–20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

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.

Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques.

Rhodamines NN: A Novel Class of Caged Fluorescent Dyes**

Caged (that is, masked) fluorescent dyes are maintained in their nonfluorescent state by the incorporation of a photochemical labile group. The photosensitive masking group or “molecular cage” can be cleaved-off by irradiation with nearUV light, thereby rendering the dye fluorescent. Caged fluorescent dyes are of enormous interest for biological imaging because they may be used, for example, for the analysis of protein dynamics, multicolor fluorescence microscopy, and far-field optical nanoscopy. o-Nitrobenzyl groups are often used as masking groups; however, the use of these dyes is limited because of their rather complex synthesis and the unwanted by-products liberated by photolysis. Herein we report on the synthesis and characterization of a novel class of caged compounds—rhodamine NN dyes, which have a 2-diazoketone (COCNN) caging group incorporated into a spiro-9H-xanthene fragment (compounds 3 and 9-R in Schemes 1 and 3, respectively). This very simple and small caging group is the core element of a new class of masked rhodamines that have remarkable properties. The rhodamine NN dyes can be easily prepared and conjugated with biomolecules, they undergo rapid uncaging under standard irradiation conditions (with wavelengths 420 nm) with formation of highly fluorescent rhodamine derivatives, and they can be used in aqueous buffers, as well as in various embedding media utilized in imaging applications. In microscopy, these novel rhodamines may be used as labels alone or in combination with conventional fluorescent dyes and switchable rhodamine spiroamides. In the latter case, they enable new imaging protocols based on the stepwise activation and detection of several fluorescent markers. The combination of the new rhodamine NN derivative (9-R) with the photochromic spiroamide of rhodamine S and a normal (uncaged) N,N,N’,N’-tetramethylrhodamine resulted in a monochoromatic multilabel imaging scheme with low cross-talk, despite using three fluorophores with very similar absorption and emission spectra. Rhodamines are very photostable and bright fluorescent dyes which can readily be chemically modified and caged. Coumarines and fluorescein have also been used as caged fluorescent dyes. As a photocleavable unit, most of these caged compounds contain a 2-nitrobenzyl group or a derivative with an alkyl or a carboxy group in the a position to the phenyl ring (at the CH2 group) and/or one or two methoxy groups in the aromatic ring. Compounds with a free carboxy group are required for bioconjugation. However, the synthesis of caged rhodamines with a free (“second”) carboxy group is difficult and their yield is low. The 2-nitrobenzyl group and its substitutes are bulky and generate toxic, colored, and highly reactive 2-nitrosobenzaldehyde or 2-nitrosobenzophenone derivatives upon photolysis. These compounds or their oligomers are expected to be poisonous to living cells, and they are also colored and interfere with optical measurements. Other modern caging groups with the required absorption in the near-UV region are also bulky, rather lipophilic, and the procedures for their synthesis and introduction are often complex. For example, 2-(N,N-dimethylamino)-5-nitrophenol was reported to give photocleavable phenyl esters. 7-Diethylamino-4-(hydroxymethyl)-2H-chromen-2-one is known to form esters which can be cleaved easily by irradiation at 412 nm. Derivatives of 8-bromo-7-hydroxyquinoline and 6-bromo-7-hydroxycoumarines have also been proposed as light-sensitive protecting groups. The photolysis of these caged compounds generates light-absorbing by-products. We set out to prepare masked fluorescent dyes without bulky caging groups. A very small 2-diazoketone fragment would be an ideal caging group, provided that it is still possible to integrate this group into the colorless form of a fluorescent dye and then restore the fluorescent state by photolysis. Rhodamines are ideal for this purpose, because they contain a carboxy group, which is known to form colorless and nonfluorescent lactones or lactams with the spiro-9H-xanthene fragment. Furthermore, this carboxy group may be transformed into a 2-diazoketone residue. For the practical realization of this caging strategy, we used rhodamine B as a model compound and performed the reaction of diazomethane with its acid chloride 1. The yellow crystalline diazoketone 3 was obtained in high yield (Scheme 1). In the course of the facile caging reaction, the positively charged C9 atom of the xanthene fragment attacks the negatively charged carbon atom of the diazomethane residue in the intermediate 2. The simultaneous abstraction of a proton furnishes the stable five-membered ring. [*] Dr. V. N. Belov, Dr. C. A. Wurm, Dr. V. P. Boyarskiy, Dr. S. Jakobs, Prof. Dr. S. W. Hell Department of NanoBiophotonics Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 G ttingen (Germany) Fax: (+49)551-201-2506 E-mail: vbelov@gwdg.de cwurm@gwdg.de shell@gwdg.de Homepage: http://www.mpibpc.gwdg.de/abteilungen/200/

Fluorescence Nanoscopy with Optical Sectioning by Two‐Photon Induced Molecular Switching using Continuous‐Wave Lasers

During the last decade far-field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous-wave laser beam. This bright marker enables strict control of on/off switching and provides single-molecule localization precision down to 15 nm in the focal plane. Two-photon induced nonlinear photoswitching of this marker with continuous-wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost-effective fluorescence nanoscopy with noninvasive optical sectioning.

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.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

Abstract A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500–630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye–ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure–property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one‐ and two‐color images of living cells with an optical resolution of 40–60 nm.

4‐Trifluoromethyl‐Substituted Coumarins with Large Stokes Shifts: Synthesis, Bioconjugates, and Their Use in Super‐Resolution Fluorescence Microscopy

Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.

Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses.

Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels (“multilevelSTED”) optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.