Rational Engineering of Photoconvertible Fluorescent Proteins for Dual‐Color Fluorescence Nanoscopy Enabled by a Triplet‐State Mechanism of Primed Conversion

Green‐to‐red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super‐resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400‐nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far‐red/near‐infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate “primed” state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create “pr”‐ (for primed convertible) variants of most known green‐to‐red pcFPs.     

Addressing the Requirements of High‐Sensitivity Single‐Molecule Imaging of Low‐Copy‐Number Proteins in Bacteria

Single‐molecule fluorescence super‐resolution imaging and tracking provide nanometer‐scale information about subcellular protein positions and dynamics. These single‐molecule imaging experiments can be very powerful, but they are best suited to high‐copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single‐molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single‐molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.     

Conversion of red fluorescent protein into a bright blue probe

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in F?rster resonance energy transfer applications.     

Far‐Red Organic Fluorophores Contain a Fluorescent Impurity

Far‐red organic fluorophores commonly used in traditional and super‐resolution localization microscopy are found to contain a fluorescent impurity with green excitation and near‐red emission. This near‐red fluorescent impurity can interfere with some multicolor stochastic optical reconstruction microscopy/photoactivated localization microscopy measurements in live cells and produce subtle artifacts in chemically fixed cells. We additionally describe alternatives to avoid artifacts in super‐resolution localization microscopy.     

Spectral Versatility of Single Reef Coral Fluorescent Proteins Detected by Spectrally‐Resolved Single Molecule Spectroscopy

We use spectrally‐resolved room temperature single molecule spectroscopy to yield insights into the occurrence and dynamics of spectral forms of single tetramers of DsRed and its variants DsRed2, Fluorescent Timer, DsRed_N42H and AG4. The red‐emitting chromophore in DsRed and all studied variants readily converts into a high quantum efficiency super‐red emitting form. We propose the existence of two super‐red forms of different quantum efficiencies. The observed emission from the green‐emitting chromophore is consistent with bulk spectroscopy. We further observe distinct new spectral forms from each variant, which we attribute to a photoinduced chemical reaction leading to a truncated form of the red‐emitting chromophore analogous to the chromophore in the visible fluorescent protein zFP538. Our results have implications for the accurate interpretation of biological and biochemical processes illuminated by fluorescent proteins as well as for choosing appropriate experimental configurations.     

Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy.     

Make them Blink: Probes for Super‐Resolution Microscopy

In recent years, a number of approaches have emerged that enable far‐field fluorescence imaging beyond the diffraction limit of light, namely super‐resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super‐resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single‐molecule compatible fluorophore can be used for super‐resolution microscopy. We present examples for super‐resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super‐resolution microscopes including AFM‐based single‐molecule assembly and DNA origami.     

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

Fluorescent proteins (FPs) are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. In this paper, we use molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variant. We employ implicit ligand sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. We also show that these pathways can be blocked or altered and barrel fluctuations can be reduced by strategic amino acid substitutions.     

Site‐Specific Labeling of Affimers for DNA‐PAINT Microscopy

Optical super‐resolution techniques allow fluorescence imaging below the classical diffraction limit of light. From a technology standpoint, recent methods are approaching molecular‐scale spatial resolution. However, this remarkable achievement is not easily translated to imaging of cellular components, since current labeling approaches are limited by either large label sizes (antibodies) or the sparse availability of small and efficient binders (nanobodies, aptamers, genetically‐encoded tags). In this work, we combined recently developed Affimer reagents with site‐specific DNA modification for high‐efficiency labeling and imaging using DNA‐PAINT. We assayed our approach using an actin Affimer. The small DNA‐conjugated affinity binders could provide a solution for efficient multitarget super‐resolution imaging in the future.     

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically‐Encoded Probes for DNA‐PAINT

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super‐resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically‐encoded self‐labeling enzymes such as SNAP‐tag and HaloTag with DNA‐PAINT microscopy. We resolve single copies of nucleoporins in the human Y‐complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.     

SLAP: Small Labeling Pair for Single‐Molecule Super‐Resolution Imaging

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small‐molecule recognition unit (Ni‐trisNTA) and the genetically encoded minimal protein His_6‐10‐tag. High‐density protein tracing by SLAP was demonstrated. This technique allows super‐resolution fluorescence imaging and fulfills the necessary sampling criteria for single‐molecule localization‐based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).     

In Situ Visualization of Block Copolymer Self‐Assembly in Organic Media by Super‐Resolution Fluorescence Microscopy

Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media.     

Super‐Chelators for Advanced Protein Labeling in Living Cells

Live‐cell labeling, super‐resolution microscopy, single‐molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N‐nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA‐based small interaction pairs described so far. Coupled to bright organic fluorophores with fine‐tuned photophysical properties, the super‐chelator probes were delivered into human cells by chemically gated nanopores. These super‐chelators permit kinetic profiling, multiplexed labeling of His₆‐ and His₁₂‐tagged proteins as well as single‐molecule‐based super‐resolution imaging.     

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

There is a persistent need for small‐molecule fluorescent labels optimized for single‐molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red‐shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed “DCDHF” fluorophores, for use in live‐cell imaging based on the push–pull design: an amine donor group and a 2‐dicyanomethylene‐3‐cyano‐2,5‐dihydrofuran (DCDHF) acceptor group, separated by a π‐rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red‐emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low‐intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small‐molecule fluorophores, which are needed for super‐resolution imaging schemes that require active control (here turning‐on) of single‐molecule emission.     

Cross‐Platform DNA Encoding for Single‐Cell Imaging of Gene Expression

Integration of imaging data across different molecular target types can provide in‐depth insight into cell physiology and pathology, but remains challenging owing to poor compatibility between target‐type‐specific labeling methods. We show that cross‐platform imaging analysis can be readily achieved through DNA encoding of molecular targets, which translates the molecular identity of various target types into a uniform in situ array of ssDNA tags for subsequent labeling with complementary imaging probes. The concept was demonstrated through multiplexed imaging of mRNAs and their corresponding proteins with multicolor quantum dots. The results reveal heterogeneity of cell transfection with siRNA and outline disparity in RNA interference (RNAi) kinetics at the level of both the mRNA and the encoded protein.     

Universal Super‐Resolution Multiplexing by DNA Exchange

Super‐resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time‐consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super‐resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.     

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe.     

Nanoscopic Visualization of Soft Matter Using Fluorescent Diarylethene Photoswitches

The in situ imaging of soft matter is of paramount importance for a detailed understanding of functionality on the nanoscopic scale. Although super‐resolution fluorescence microscopy methods with their unprecedented imaging capabilities have revolutionized research in the life sciences, this potential has been far less exploited in materials science. One of the main obstacles for a more universal application of super‐resolved fluorescence microscopy methods is the limitation of readily available suitable dyes to overcome the diffraction limit. Here, we report a novel diarylethene‐based photoswitch with a highly fluorescent closed and a nonfluorescent open form. Its photophysical properties, switching behavior, and high photostability make the dye an ideal candidate for photoactivation localization microscopy (PALM). It is capable of resolving apolar structures with an accuracy far beyond the diffraction limit of optical light in cylindrical micelles formed by amphiphilic block copolymers.     

Masked Rhodamine Dyes of Five Principal Colors Revealed by Photolysis of a 2‐Diazo‐1‐Indanone Caging Group: Synthesis,Photophysics, and Light Microscopy Applications

Caged rhodamine dyes (Rhodamines NN) of five basic colors were synthesized and used as “hidden” markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2‐diazo‐1‐indanone group can be irreversibly photoactivated, either by irradiation with UV‐ or violet light (one‐photon process), or by exposure to intense red light (λ～750 nm; two‐photon mode). All dyes possess a very small 2‐diazoketone caging group incorporated into the 2‐diazo‐1‐indanone residue with a quaternary carbon atom (C‐3) and a spiro‐9H‐xanthene fragment. Initially they are non‐colored (pale yellow), non‐fluorescent, and absorb at λ=330–350 nm (molar extinction coefficient (ε)≈10⁴ M^?1 cm^?1) with a band edge that extends to about λ=440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511–633 and 525–653 nm, respectively). The unmasked dyes are highly colored and fluorescent (ε= 3–8×10⁴ M^?1 cm^?1 and fluorescence quantum yields (?)=40–85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water‐soluble caged red‐emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino‐reactive N‐hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375–420 nm light or intense red light (λ=775 nm). Protein conjugates with optimal degrees of labeling (3–6) were prepared and uncaged with λ=405 nm light in aqueous buffer solutions (?=20–38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10–40 % of the non‐fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a “dark” non‐emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super‐resolution optical microscopy is exemplified.     

Identification of sites within a monomeric red fluorescent protein that tolerate peptide insertion and testing of corresponding circular permutations

In recent years the class of known fluorescent proteins (FPs) has dramatically expanded as an ever-increasing numbers of variants and homologs of the green fluorescent protein (GFP) from Aequorea jellyfish have been either engineered in the lab or discovered in other marine organisms. The red fluorescent protein (RFP) from Discosoma coral (also known as dsFP583 and DsRed) has proven to be a particularly fruitful progenitor of variants with biochemical and spectroscopic properties conducive to applications in live cell imaging. We have investigated the tolerance of an engineered monomeric descendent of Discosoma RFP, known as mCherry, towards peptide insertion and circular permutation. Starting from a random library of insertion variants, we identified six genetically distinct sites localized in three different loops where a sequence of five residues could be inserted without abolishing the ability of the protein to form its intrinsic red fluorescent chromophore. For each of these insertion variants, a corresponding circular permutation variant was created in which the original N- and C-termini were connected by a six-residue linker and new termini were introduced at the site of the insertion. All six circular permutation variants had significantly diminished brightness relative to the analogous insertion variants. The most promising circular permutation variant has termini at the position corresponding to residue 184 of mCherry and retains 37% of the intrinsic fluorescent brightness of mCherry. These circularly permuted variants may serve as the foundation for construction of genetically encoded Ca2+ sensors analogous to the previously reported camgaroo, pericam and G-CaMP sensors based on variants of Aequorea GFP.