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 analytes 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 non-cognate analytes, in a process that resembles transport by nuclear pores.

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.

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.

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications.

Smart fluorophores", such as reversibly switchable fluorescent proteins (RSFPs), 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 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 multi-modal 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.KEYWORDS: fluorescent proteins, reversible photoswitching, super-resolution fluorescence microscopy, SOFI, RESOLFT, crystal structure determination, rsEGFP, superfolder Fluorescent proteins (FPs) enable the minimally-invasive labeling of intracellular structures in live systems. 1 The discovery and development of "smart photoactive FPs", 2,3 with features such as irreversible photoactivation and photoconversion, or reversible photoswitching, allowed the development of diffraction-unlimited imaging techniques such as (f)PALM 4,5 ((fluorescence) photoactivated localization microscopy), RESOLFT 6 (reversible saturable optical fluorescence transitions) and (pc)SOFI 7,8 ((photochromic) stochastic optical fluctuation imaging). These techniques strongly rely on the performance of the fluorophores and considerable efforts have therefore been dedicated to create optimized "smart labels". 9 This is exemplified by the continuous optimization and diversification of the EosFP family, 10-15 or the development of Dronpa 16 mutants with different or added photophysical properties. [17][18][19][20][21][22] Probes that combine multiple "smart" behaviors have also been engineered. [23][24][25] On the whole, however, the general acceptance of the FP-based "smart labels" has not quite risen up to the high expectations set by the many applications they enable. In some cases this is due to concerns surrounding the biological compatibility of the labels, meaning that the label may interfere with the functioning of the syst...

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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.

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