Genetically encoded green calcium indicators (GECIs) are broadly used for visualizing calcium transients in living cells. Among the diverse family of green GECIs, the Troponin C-based family offers potential advantages, including reduced calcium buffering, smaller molecular size, linear calcium response, and low cytotoxicity. However, the Troponin C-based GECIs with inverted calcium response are less developed compared to other popular GECIs, including GCaMPs and GECOs, and, as a consequence, have several drawbacks related to low dynamic range, brightness, photostability, and calcium ion sensitivity. To address these limitations, we developed a novel GECI, called icBTnC2, which incorporates Troponin C as a sensing moiety and the new bright photostable green FP mBaoJin as a reporting moiety. icBTnC2 demonstrated an inverted fluorescent response to calcium ion binding with a Kd of 62 nM. In terms of fluorescence contrast and calcium ion affinity in vitro, icBTnC2 was comparable to the best widely used calmodulin-based GECIs from the GCaMP family. icBTnC2 demonstrated superior photostability under wide-field fluorescence microscopy exhibiting 5.5-, 4.8-, 3.2-, 2.9-, and 1.3-fold higher photobleaching half-time compared to iYTnC2, mEGFP, NCaMP7, jGCaMP8f, and mBaoJin, respectively. The icBTnC2 indicator was benchmarked against other GECIs, such as jGCaMP8f, NCaMP7, iYTnC2, and R-GECO1, for visualization of calcium transients in mammalian cells and primary neuron cultures, and tested for calcium-dependent changes in fluorescence lifetime. Finally, we solved the crystal structure of the icBTnC2 indicator at 1.55 Å resolution in the calcium-bound state and, using directed mutagenesis, proposed the molecular basis of its fluorescent response to calcium ion binding.

The diverse spectral and photochemical properties of fluorescent proteins enable imaging applications ranging from organelle labeling to super-resolution and multiplexed live-cell microscopy. Here, we report three far-red fluorescent proteins, named mfRFP, mfRFP-A, and mCardinal-A, that share similar fluorescence spectra (excitation/emission ∼600/660 nm) but exhibit distinct photobleaching rates. Exploiting differential photostability, we performed per-pixel unmixing of three proteins simultaneously using temporal domain multiplexing (TDM), acquiring BrainBow-like images of cellular populations and resolving subcellular structures in 3D within a single imaging channel, without hardware modifications. We established quantitative criteria for selecting FP pairs that support efficient TDM unmixing and benchmarked TDM against fluorescence lifetime- and photobleaching kinetics-based alternatives. The most photostable variant, mfRFP, was further validated for STED super-resolution imaging of structural proteins in mammalian cells and for neuroimaging in mice, zebrafish, and C. elegans.

Genetically encoded voltage indicators (GEVIs) enable noninvasive, high-speed monitoring of electrical activity but are constrained by limited brightness and rapid photobleaching under continuous illumination. Here, we present Vega, a highly photostable green fluorescence GEVI with both high sensitivity (ΔF/F = -33% per 100 mV) and fast response (1.34 ms). Under one-photon excitation at 1 W/cm[2][1], Vega exhibits more than 20-fold slower photobleaching than the spectrally similar GEVI, Ace-mNeon2. In acute mouse brain slice, Vega enabled wide-field high-fidelity recording of action potentials from 51 neurons simultaneously. In pancreatic islets, it revealed heterogeneous β-cell activation and intercellular coupling in response to glucose elevation. Finally, one-photon imaging in awake mice demonstrated stable cortical voltage mapping in vivo . Vega thus overcomes the longstanding photostability-performance trade-off, enabling chronic, high-fidelity voltage imaging across preparations. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-2

The PSmOrange and PSmOrange2 fluorescent proteins undergo irreversible photoconversion from the orange to far-red form under blue light, which makes them probes of choice for protein tracking and single-molecule super-resolution imaging. However, both proteins exhibit noticeable photoconversion under 550-570 nm light used for excitation of their orange form, which complicates applications of these photoconvertible FPs in cell imaging experiments. Here, we report the next-generation PSmOrange variant, called PSmOrange3, which is characterized by minimal photoconversion under 550-570 nm light and high photoconversion contrast. PSmOrange3 undergoes efficient photoconversion from the orange (Ex/Em at 550 nm/564 nm) to far-red form (Ex/Em at 614 nm/655 nm) with 430-470 nm violet-blue light of moderate power density (3-180 mW/mm2) in a native cellular environment. The molecular brightness of orange and far-red forms of PSmOrange3 was 1.2- and 1.4-fold brighter than that of PSmOrange2. In addition, PSmOrange3 had a substantially higher photostability of the orange form but a little less photostability of the far-red form. We solved the crystal structure of PSmOrange3 at a 2.8 Å resolution, which confirmed its monomeric state and revealed the role of the introduced mutations in the properties of PSmOrange3. Using mass spectrometry we revealed the chemical structure of the PSmOrange3 chromophore before and after photoconversion. PSmOrange3 was properly localized with different protein fusions and photoconverted from the orange to far-red state inside live and fixed mammalian cells without exogenously supplied oxidants. Among all proteins of the PSmOrange series, both forms of PSmOrange3 were the brightest in the reducing environment of the mitochondrial lumen. PSmOrange3 photoconverted efficiently with blue light and almost did not photoconvert with green light, which allows investigators to excite its orange form and photoconvert it to the far-red form with different light. We demonstrated the applicability of PSmOrange3 for photoactivated localization microscopy (PALM) of tubulin microtubules using 488-nm photoconversion, achieving mean localization precision per single-molecule event of 24.6 and 23.3 nm in fixed and live mammalian cells, respectively. We believe that PSmOrange3 can represent a suitable alternative to the PSmOrange and PSmOrange2 proteins and will be a valuable addition to the repertoire of available photoconvertible fluorescent proteins.

Potassium ion (K+) dynamics are vital for various biological processes. However, the limited availability of detection tools for tracking intracellular and extracellular K+ has impeded a comprehensive understanding of the physiological roles of K+ in intact biological systems. In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2, through a combination of directed evolution in Escherichia coli and subsequent optimization in mammalian cells. RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively. We employed RGEPOs for real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and the awake mouse in both intracellular and extracellular environments. Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice. Furthermore, molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features. Thus, RGEPOs represent a significant advancement in potassium imaging, providing enhanced tools for real-time visualization of K+ dynamics in various cell types and cellular environments.

Recent advancements in genetically encoded calcium indicators, particularly those based on green fluorescent proteins, have optimized their performance for monitoring neuronal activities in a variety of model organisms. However, progress in developing red-shifted GECIs, despite their advantages over green indicators, has been slower, resulting in fewer options for end users. In this study, we explored topological inversion and soma-targeting strategies, which are complementary to conventional mutagenesis, to re-engineer a red genetically encoded calcium indicator, FRCaMP, for enhanced in vivo performance. The resulting sensors, FRCaMPi and soma-targeted FRCaMPi (SomaFRCaMPi), exhibit up to 2-fold higher dynamic range and peak ΔF/F0 per single AP compared to widely used jRGECO1a in neurons both in culture and in vivo. Compared to jRGECO1a and FRCaMPi, SomaFRCaMPi reduces erroneous correlation of neuronal activity in the brains of mice and zebrafish by two- to 4-fold due to diminished neuropil contamination without compromising the signal-to-noise ratio. Under wide-field imaging in primary somatosensory and visual cortices in mice with high labeling density (80-90%), SomaFRCaMPi exhibits up to 40% higher SNR and decreased artifactual correlation across neurons. Altogether, SomaFRCaMPi improves the accuracy and scale of neuronal activity imaging at single-neuron resolution in densely labeled brain tissues due to a 2-3-fold enhanced automated neuronal segmentation, 50% higher fraction of responsive cells, up to 2-fold higher SNR compared to jRGECO1a. Our findings highlight the potential of SomaFRCaMPi, comparable to the most sensitive soma-targeted GCaMP, for precise spatial recording of neuronal populations using popular imaging modalities in model organisms such as zebrafish and mice.

Recent advancements in genetically encoded calcium indicators, particularly those based on green fluorescent proteins, have optimized their performance for monitoring neuronal activities in a variety of model organisms. However, progress in developing red-shifted GECIs, despite their advantages over green indicators, has been slower, resulting in fewer options for end-users. In this study, we explored topological inversion and soma-targeting strategies, which are complementary to conventional mutagenesis, to re-engineer a red genetically encoded calcium indicator, FRCaMP, for enhanced in vivo performance. The resulting sensors, FRCaMPi and soma-targeted FRCaMPi (SomaFRCaMPi), exhibit up to 2-fold higher dynamic range and peak ΔF/F0 per single AP compared to widely used jRGECO1a in neurons in culture and in vivo. Compared to jRGECO1a and FRCaMPi, SomaFRCaMPi reduces erroneous correlation of neuronal activity in the brains of mice and zebrafish by two- to four-fold due to diminished neuropil contamination without compromising the signal-to-noise ratio. Under wide-field imaging in primary somatosensory and visual cortex in mice with high labeling density (80-90%), SomaFRCaMPi exhibits up to 40% higher SNR and decreased artifactual correlation across neurons. Altogether, SomaFRCaMPi improves the accuracy and scale of neuronal activity imaging at single-neuron resolution in densely labeled brain tissues due to a 2-3-fold enhanced automated neuronal segmentation, 50% higher fraction of responsive cells, up to 2-fold higher SNR compared to jRGECO1a. Our findings highlight the potential of SomaFRCaMPi, comparable to the most sensitive soma-targeted GCaMP, for precise spatial recording of neuronal populations using popular imaging modalities in model organisms such as zebrafish and mice.

Genetically encoded voltage indicators (GEVIs) are powerful tools for monitoring neuronal activity, but their application, particularly for long-term recordings in vivo , is often limited by photobleaching under the required high illumination intensities. This constraint restricts the total duration of continuous or trial-based experiments, crucial for studying processes like synaptic plasticity or circuit dynamics during behavior. Here, we introduce ElectraON and ElectraOFF, a pair of green fluorescent eFRET-based GEVIs engineered by incorporating a photostability-enhanced derivative of the bright monomeric fluorescent protein mBaoJin with Ace opsin variants. Critically, Electras demonstrate over 6-fold improved photostability compared to state-of-the-art eFRET GEVIs, pAce, and Ace-mNeon2, under one-photon illumination, while characterized by bright green fluorescence, millisecond kinetics, and good membrane localization. This enhanced stability translates to a 3-to >10-fold extension in functional recording duration, maintaining reliable spike detection in both cultured neurons in vitro and sparsely labeled neurons in the awake mouse cortex in vivo . We demonstrated sustained in vivo recordings exceeding 30 minutes, with instances surpassing one hour. Furthermore, Electras show functionality under scanless two-photon excitation in cultured cells. These highly photostable indicators significantly extend the temporal window for voltage imaging, broadening the scope of accessible biological questions. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, https://ror.org/01h0zpd94, 32171093, 323B200334

The diverse spectral and photochemical properties of fluorescent proteins enable a variety of imaging applications in cell biology, ranging from cellular and organelle labeling to super-resolution microscopy and multiplexed live cell imaging. Here, we report a set of novel far-red fluorescent proteins, named mfRFP, mfRFP-A, and mCardinal-A, which are characterized by similar fluorescence spectra with excitation/emission at ∼600/660 nm while exhibiting distinct photobleaching rates. Differences in photostability allowed us to perform per-pixel unmixing of the three far-red FPs imaged simultaneously by employing a recently introduced temporal domain multiplexing approach. We demonstrated the application of the temporal domain multiplexing approach with different combinations of far-red fluorescent proteins possessing nearly identical emission spectra by acquiring BrainBow-like images of cellular populations and distinguishing subcellular structures in mammalian cells using a single imaging channel without applying any hardware modifications to the conventional microscope. Unlike previous temporal domain multiplying approaches employing photophysical properties of fluorescent proteins, the current approach is a wide range of microscopy modalities, including 3D imaging with a spinning disk and point scanning confocal microscopy. The most photostable fluorescent protein in the set, mfRFP, was further benchmarked against spectrally similar FPs and applied for super-resolution imaging of structural proteins in mammalian cells and for neuroimaging of model organisms, including mice, zebrafish, and C. elegans . ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, https://ror.org/01h0zpd94, 32171093

Genetically encoded fluorescent biosensors became indispensable tools for biological research, enabling real-time observation of physiological processes in live cells. Recent protein engineering efforts have resulted in the generation of a large variety of fluorescent biosensors for a wide range of biologically relevant processes, from small ions to enzymatic activity and signaling pathways. However, biosensors for imaging sulfate ions, the fourth most abundant physiological anion, in mammalian cells are still lacking. Here, we report the development and characterization of a green fluorescent biosensor for sulfate named Thyone. Thyone, derived through structure-guided design from bright green fluorescent protein mNeonGreen, exhibited a large negative fluorescence response upon subsecond association with sulfate anion with an affinity of 11 mM in mammalian cells. By integrating mutagenesis analyses with molecular dynamics simulations, we elucidated the molecular mechanism of sulfate binding and revealed key amino acid residues responsible for sulfate sensitivity. High anion selectivity and sensitivity of Thyone allowed for imaging of sulfate anion transients mediated by sulfate transporter heterologously expressed in cultured mammalian cells. We believe that Thyone will find a broad application for assaying the sulfate transport in mammalian cells via anion transporters and exchangers. A genetically encoded green fluorescent sensor, Thyone, enables real-time imaging of sulfate anion dynamics in mammalian cells to probe the sulfate transport activity of the SLC26A2 protein.

Potassium ion (K+) dynamics are vital for various biological processes. However, the limited availability of detection tools for tracking intracellular and extracellular K+ has impeded a comprehensive understanding of the physiological roles of K+ in intact biological systems. In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2, through a combination of directed evolution in E. coli and subsequent optimization in mammalian cells. RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 3.55 mM and 14.81 mM in HEK293FT cells, respectively. We employed RGEPOs for real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and the awake mouse in both intracellular and extracellular environments. Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice. Furthermore, molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features. Thus, RGEPOs represent a significant advancement in potassium imaging, providing enhanced tools for real-time visualization of K+ dynamics in various cell types and cellular environments. ### Competing Interest Statement The authors have declared no competing interest.

Branched-chain amino acids (BCAAs) play an important role in the functioning of mammalian cells and the central nervous system. However, available genetically encoded indicators for BCAAs are based on Forster resonance energy transfer and have a limited dynamic range. We developed a single fluorescent protein-based sensor for BCAAs, called NeIle, which is composed of circularly permutated mNeonGreen protein inserted into the leucine-isoleucine-valine binding protein (LIVBP) from Escherichia coli bacteria. In solution, the NeIle indicator displayed a positive fluorescence response to adding isoleucine, leucine, and valin amino acids with high Delta F/F dynamic ranges of 27-, 19-, and 11-fold and the corresponding affinity values of 5.0, 2.9, and 75 mM, respectively. The spectral and biochemical properties of the NeIle indicator were characterized in solution. We characterized the brightness of the NeIle indicator in living mammalian cells, including cultured neurons. Using the NeIle indicator, we successfully visualized the dynamics of isoleucine transients in different organelles of mammalian cells. We obtained and analyzed the X-ray crystal structure of the NeIle indicator in an isoleucine-bound state. Structure-guided directed mutagenesis of the NeIle indicator revealed the basis of its fluorescence response and selectivity to isoleucine.

Heavy metals, particularly mercury, rank as some of the most hazardous systemic toxicants known to cause multiple organ damage, even at lower levels of exposure. Its detection in the environment and in the live cells is an actual task. Here, we engineered a novel genetically encoded fluorescent NMT indicator for mercury ions by inserting the metallothionein II domain from rat liver into the bright green-yellow fluorescent protein mNeonGreen, followed by directed molecular evolution of the resulting sensor prototype in bacteria. In solution, the NMT indicator was 1.7-fold brighter than the standard eGFP fluorescent protein and responded to the addition of even 10-18-10-19 M mercury ions by quenching fluorescence with a 5-fold fluorescence response and extremely high affinity to mercury ions characterized by the Kd value of 0.50 +/- 0.05 aM. We also characterized the selectivity of the NMT indicator to other metal cations. In cultured mammalian cells, the NMT indicator detected even an extracellular concentration of 0.1 fM mercury ions and achieved a 5.9-fold change in Delta F/F fluorescence intensity.

The detection of mercury ions is an important task in both environmental monitoring and cell biology research. However, existing genetically encoded sensors for mercury ions have certain limitations, such as negative fluorescence response, narrow dynamic range, or the need for cofactor supplementation. To address these limitations, we have developed novel sensors by fusing a circularly permutated version of the mNeonGreen green fluorescent protein with the merP mercury-binding protein from Gram-negative bacteria Shigella flexneri. The developed NeMeHg and iNeMeHg sensors responded to mercury ions with positive and negative fluorescence changes, respectively. We characterized their properties in vitro. Using the developed biosensors, we were able to successfully visualize changes in mercury ion concentration in mammalian cultured cells.

High brightness and photostability of StayGold make it a particularly attractive probe for long-term live cell imaging. However, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and X-ray structures of a monomeric variant of StayGold (mBaoJin), which preserves the beneficial properties of its precursor while serving as a tag for structural proteins and membranes. We compare mBaoJin to other state-of-art GFPs and utilize it for super-resolution long-term live cell imaging and expansion microscopy.

Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising alternative to control the gene expression at the spatiotemporal transcriptomic level without complete elimination. The innovative CRISPR-Cas RNA-targeting systems changed the conception of biosensing systems and also allowed the RNA effectors to be used in various applications; for example, genomic editing, effective virus diagnostic tools, biomarkers, transcription regulations. In this review, we discussed the current state-of-the-art of specific CRISPR-Cas systems known to bind and cleave RNA substrates and summarized potential applications of the versatile RNA-targeting systems.

Engineered light, oxygen, and voltage (LOV)-based proteins are able to fluoresce without oxygen requirement due to the autocatalytic incorporation of exogenous flavin as a chromophore thus allowing for live cell imaging under hypoxic and anaerobic conditions. They were also discovered to have high sensitivity to transition metal ions and physiological flavin derivatives. These properties make flavin-binding fluorescent proteins (FPs) a perspective platform for biosensor development. However, brightness of currently available flavin-binding FPs is limited compared to GFP-like FPs creating a need for their further enhancement and optimization. In this study, we applied a directed molecular evolution approach to develop a pair of flavin-binding FPs, named miniGFP1 and miniGFP2. The miniGFP proteins are characterized by cyan-green fluorescence with excitation/emission maxima at 450/499 nm and a molecular size of ∼13 kDa. We carried out systematic benchmarking of miniGFPs in Escherichia coli and cultured mammalian cells against spectrally similar FPs including GFP-like FP, bilirubin-binding FP, and bright flavin-binding FPs. The miniGFPs proteins exhibited improved photochemical properties compared to other flavin-binding FPs enabling long-term live cell imaging. We demonstrated the utility of miniGFPs for live cell imaging in bacterial culture under anaerobic conditions and in CHO cells under hypoxia. The miniGFPs’ fluorescence was highly sensitive to Cu(II) ions in solution with Kd values of 67 and 68 nM for miniGFP1 and miniGFP2, respectively. We also observed fluorescence quenching of miniGFPs by the reduced form of Cu(I) suggesting its potential application as an optical indicator for Cu(I) and Cu(II). In addition, miniGFPs showed the ability to selectively bind exogenous flavin mononucleotide demonstrating a potential for utilization as a selective fluorescent flavin indicator. Altogether, miniGFPs can serve as a multisensing platform for fluorescence biosensor development for in vitro and in-cell applications.