Quantitative determination of the full switching cycle of photochromic fluorescent proteins.

In this study, we develop a general analytical model of the photochromism of fluorescent proteins and apply it to spectroscopic measurements performed on six different labels. Our approach provides quantitative explanations for phenomena such as the existence of positive and negative switching, limitations in the photochromism contrast, and the fact that initial switching cycles may differ from subsequent ones. It also allows us to perform the very first measurement of all four isomerization quantum yields involved in the switching process.

Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry.

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4 °C and a few days at 37 °C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate. X-ray crystallography and mass spectrometry reveal that this is caused by the cleavage of the p-hydroxyphenyl ring from the chromophore and the formation of two novel types of cyclic structures at the remaining chromophore moiety. Overall, our work sheds light on a new process occurring within fluorescent proteins, further adding to the chemical diversity and versatility of these molecules.

Absolute measurement of cellular activities using photochromic single-fluorophore biosensors and intermittent quantification.

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.

The HaloTag as a general scaffold for far-red tunable chemigenetic indicators.

Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

Orthogonal fluorescent chemogenetic reporters for multicolor imaging.

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein-protein interactions by live-cell fluorescence microscopy.

Genetically encoded biosensors based on innovative scaffolds.

Genetically encoded biosensors are indispensable tools for visualizing the spatiotemporal dynamics of analytes or processes in living cells in vitro and in vivo. Their widespread adaptation has gone hand in hand with the development of sensors for new analytes or processes and improved functionality and robustness. In this review, we highlight some of the recent advances in genetically encoded biosensor development, with a special focus on novel and innovative scaffolds that will lead to new possibilities in the future.

Freeze-frame imaging of synaptic activity using SynTagMA.

Information within the brain travels from neuron to neuron across billions of synapses. At any given moment, only a small subset of neurons and synapses are active, but finding the active synapses in brain tissue has been a technical challenge. Here we introduce SynTagMA to tag active synapses in a user-defined time window. Upon 395-405 nm illumination, this genetically encoded marker of activity converts from green to red fluorescence if, and only if, it is bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we show how to identify and track the fluorescence of thousands of individual synapses in an automated fashion. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior.

SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data.

Super-resolution fluorescence imaging techniques allow optical imaging of specimens beyond the diffraction limit of light. Super-resolution optical fluctuation imaging (SOFI) relies on computational analysis of stochastic blinking events to obtain a super-resolved image. As with some other super-resolution methods, this strong dependency on computational analysis can make it difficult to gauge how well the resulting images reflect the underlying sample structure. We herein report SOFIevaluator, an unbiased and parameter-free algorithm for calculating a set of metrics that describes the quality of super-resolution fluorescence imaging data for SOFI. We additionally demonstrate how SOFIevaluator can be used to identify fluorescent proteins that perform well for SOFI imaging under different imaging conditions.

A comprehensive dataset of image sequences covering 20 fluorescent protein labels and 12 imaging conditions for use in super-resolution imaging.

Super-resolution fluorescence microscopy techniques allow imaging fluorescently labelled structures with a resolution that surpasses the diffraction limit of light (approx. 200nm). The quality and, thus, reliability of each of these techniques is strongly dependent on (1) the quality of the optics, (2) the fitness of the specific fluorescent label for the given technique and (3) the algorithms being used. Of these, the fitness of the labels is most subjective, as fitness metrics are scarce, and generating samples with different labels and imaging them is laborious. This prevent rigorous fitness assessment of fluorescent labels. We have developed a mathematical framework for assessing the quality of SOFI data [1], [2], which we used to assess the fitness of 20 different fluorescent protein labels for SOFI imaging. Here, we report this dataset of 2240 image sequences, representing 10 fields of view each of transfected Cos7 cells expressing each of the 20 different fluorescent proteins under 4-12 imaging conditions. The labels span the visible spectrum and include non-photo-transforming and photo-transforming fluorescent proteins. The imaging conditions consist of 4 different excitation powers, each with three different powers of 405 nm light added (except for the blue labels that are excited with 405 nm light). Though this data was in essence generated to assess which labels are best suited for SOFI imaging, it can be used as a benchmark for further development of the SOFI algorithm, or for the development of other super-resolution imaging modalities that benefit from similar input data.

Improved methods for marking active neuron populations.

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

Model-free uncertainty estimation in stochastical optical fluctuation imaging (SOFI) leads to a doubled temporal resolution.

Stochastic optical fluctuation imaging (SOFI) is a super-resolution fluorescence imaging technique that makes use of stochastic fluctuations in the emission of the fluorophores. During a SOFI measurement multiple fluorescence images are acquired from the sample, followed by the calculation of the spatiotemporal cumulants of the intensities observed at each position. Compared to other techniques, SOFI works well under conditions of low signal-to-noise, high background, or high emitter densities. However, it can be difficult to unambiguously determine the reliability of images produced by any superresolution imaging technique. In this work we present a strategy that enables the estimation of the variance or uncertainty associated with each pixel in the SOFI image. In addition to estimating the image quality or reliability, we show that this can be used to optimize the signal-to-noise ratio (SNR) of SOFI images by including multiple pixel combinations in the cumulant calculation. We present an algorithm to perform this optimization, which automatically takes all relevant instrumental, sample, and probe parameters into account. Depending on the optical magnification of the system, this strategy can be used to improve the SNR of a SOFI image by 40% to 90%. This gain in information is entirely free, in the sense that it does not require additional efforts or complications. Alternatively our approach can be applied to reduce the number of fluorescence images to meet a particular quality level by about 30% to 50%, strongly improving the temporal resolution of SOFI imaging.

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy.

"Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.

Molecular Dynamic Indicators of the Photoswitching Properties of Green Fluorescent Proteins.

Reversibly photoswitchable fluorescent proteins (RSFPs) are highly useful probes for a range of applications including diffraction-unlimited fluorescence microscopy. It was previously shown that reversible photoswitching not only involves cis-trans isomerization and protonation-deprotonation of the chromophore but also results in a marked difference in ß-barrel flexibility. In this work, we performed flexibility profiling and functional mode analysis (FMA) using molecular dynamics calculations to study how the flexibility of the RSFP ß-barrel influences the photoswitching properties of several fluorescent proteins. We also used Partial Least-Squared (PLS) FMA to detect promising mutation sites for the modulation of photoswitching properties of RSFPs. Our results show that the flexibility of RSFP does depend on its state with a systematically higher flexibility in the dark state compared to the bright state. In particular our method highlights the importance of Val157 in Dronpa, which upon mutation yields a striking difference in the collective motions of the two mutants. Overall, we show that PLS-FMA yields information, complementary to static structures, that can guide the rational design of fluorescent proteins.

Diffraction-unlimited fluorescence microscopy of living biological samples using pcSOFI.

The complex microscopic nature of many live biological processes is often obscured by the diffraction limit of light, requiring diffraction-unlimited fluorescence microscopy to resolve them. Because of the vast range of different processes that can be studied, sub-diffraction imaging should work efficiently under many different conditions. Photochromic stochastic optical fluctuation imaging (pcSOFI) is a recent addition to the field of diffraction-unlimited fluorescence microscopy. This robust and versatile method employs a statistical analysis of random fluctuations in the emission of single labels, in this case reversibly switchable fluorescent proteins (RSFPs), to retrieve super-resolution information. Added to the resolution enhancement, pcSOFI also offers contrast enhancement and background reduction in a practical and convenient way. Here, we describe the necessary steps to obtain diffraction-unlimited images, including multicolor and three-dimensional imaging, and highlight the advantages of pcSOFI together with the circumstances under which pcSOFI can be favorably applied.

PcSOFI as a smart label-based superresolution microscopy technique.

Stochastic optical fluctuation imaging (SOFI) is a superresolution imaging technique that uses the flickering of fluorescent labels to generate a microscopic image with a resolution better than what the diffraction limit allows. Its adaptation towards fluorescent protein-labeled samples (called photoconversion SOFI or pcSOFI) allows for a straightforward and easily accessible way of generating superresolution images. In this protocol, we will discuss how so-called "smart labels," and specifically the reversibly switchable fluorescent proteins, have opened doors towards superresolution imaging in general and we provide a protocol on how to perform pcSOFI on HeLa cells expressing human ß-actin labeled with the reversibly photoswitchable fluorescent protein Dronpa.

Green-to-red photoconvertible Dronpa mutant for multimodal super-resolution fluorescence microscopy.

Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.

Revealing the excited-state dynamics of the fluorescent protein Dendra2.

Green-to-red photoconversion is a reaction that occurs in a limited number of fluorescent proteins and that is currently mechanistically debated. In this contribution, we report on our investigation of the photoconvertible fluorescent protein Dendra2 by employing a combination of pump-probe, up-conversion and single photon timing spectroscopic techniques. Our findings indicate that upon excitation of the neutral green state an excited state proton transfer proceeds with a time constant of 3.4 ps between the neutral green and the anionic green states. In concentrated solution we detected resonance energy transfer (25 ps time constant) between green and red monomers. The time-resolved emission spectra suggest also the formation of a super-red species, first observed for DsRed (a red fluorescent protein from the corallimorph species Discosoma) and consistent with peculiar structural details present in both proteins.

Structural basis for the influence of a single mutation K145N on the oligomerization and photoswitching rate of Dronpa.

The crystal structure of the on-state of PDM1-4, a single-mutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 Å resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain ß-strands caused by oligomerization along the AC interface.

An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry.

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at -50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.