Circularly permuted green fluorescent protein (cpGFP)-based high-performance cAMP sensors have enabled real-time monitoring of cAMP dynamics with high spatiotemporal resolution in living animals. However, their utility is hampered by significant spectral overlap with other green/yellow fluorescent indicators and blue/cyan light-activated optogenetic actuators, limiting their compatibility in multiplexed imaging applications. While existing red cAMP sensors offer great spectral separation, they often suffer from a limited dynamic range ( < 1.5-fold in HEK293T cells), low cellular brightness, aggregation, or significant blue-light-induced photoactivation. Here, we report R-Flamp1, a red cAMP sensor with a large dynamic range ( > 10-fold in HEK293T cells), enhanced cellular brightness, appropriate cAMP affinity (Kd ~1.9 μM), subsecond response kinetics, and minimal photoactivation under blue or cyan light exposure. Using R-Flamp1, we visualized region-specific cAMP dynamics, and when paired with green indicators, revealed differential activation patterns between cAMP and neuromodulators or calcium using two-photon imaging and fiber photometry during various behaviors. These findings provide valuable insights into the role of cAMP signaling in complex behaviors.

Calpain-1, a calcium-dependent cysteine protease, plays a pivotal role in various biological functions. However, real-time monitoring of its activity has been limited by the lack of high-performance calpain sensors. Here, we report the development of iFoCAL, a sensitive and specific fluorescence resonance energy transfer (FRET) sensor for calpain-1, which employs mClover3 and mScarletX, a newly engineered green-red FRET pair with weak interactions. Using iFoCAL, we revealed distinct spatiotemporal patterns of calpain-1 activation: gradual and global activation in whole cells during endoplasmic reticulum (ER) calcium release, fast and focal activation in axons during mechanical stress, and region-specific activation in a Parkinson's disease model. These findings provide the first direct evidence of distinct calpain-1 dynamics across diverse cellular contexts.

Large-scale imaging of multiple dynamic behaviors and quantitative neurochemical concentrations with high spatiotemporal resolution is essential for understanding complex brain functions. Two-photon microscopy (TPM) is ideally suited for in vivo brain function imaging because of its high resolution and deep tissue penetration. However, conventional TPM is limited by a restricted field-of-view (FOV), an inherent trade-off between the imaging area and temporal resolution, and an insufficient amount of information obtained using only intensity recording. Here, we propose large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter and quantitative brain function imaging, with a 3 × 3 mm2 FOV, a uniform lateral resolution of 0.7 μm, and a FLIM throughput of up to 15.73 megapixels/s (512 × 512 pixels, 30 Hz, two regions). We extend the FOV by breaking the limit of commercial objectives with an effective adaptive optics strategy. To alleviate the trade-off between the imaging area and temporal resolution, we use a temporal multiplexing system that enables simultaneous and flexible two-region imaging across the large FOV. Furthermore, we develop a field programmable gate array module to demultiplex fluorescence signals from different regions and perform high-throughput, two-region FLIM. We demonstrate the superior performance of LD-2P-FLIM by simultaneous monitoring of neural activities across multiple cortical areas, synchronous recording of neurovascular coupling under both physiological and pathological conditions, long-term observation of the microglial response to local neuron injury, and quantitative imaging of calcium concentrations across a large neuronal population in vivo.

Rapid escape from visual threats is essential for survival, yet the mechanisms establishing its “permissive state” remain poorly understood. Here, we demonstrate that osteocalcin (OCN), a bone-derived hormone, permits rapid visual escape by enhancing the excitability of a ventral tegmental area (VTA) GABAergic neuron subpopulation through the OCN-G Protein-Coupled Receptor 37 (GPR37)-cAMP-TWIK-related halothane-inhibited potassium channel (THIK-1) pathway. Loss of OCN, loss of its receptor GPR37, or conditional deletion of GPR37 in VTA GABAergic or glutamatergic neurons delays escape responses, while reconstituting OCN-GPR37 signaling in the VTA restores normal behavior. Single-cell transcriptomics and electrophysiology reveal that OCN suppresses THIK-1 potassium currents via GPR37-mediated cyclic AMP (cAMP) reduction, thereby increasing neuronal excitability. These findings reveal a novel mechanism through which a bone-derived hormone modulates the excitability of co-releasing neurons to facilitate rapid escape, offering new insights into the regulation of survival behaviors by the bone-brain metabolic axis.

High-resolution optical imaging of the cerebral cortex is severely hampered by strong light scattering induced by the opaque skull, posing a major obstacle to deciphering brain structure and function in vivo. Skull optical clearing, a technique that uses chemical cocktails to render the skull transparent, provides direct optical access to the cerebral cortex through the skull. However, existing chemical clearing techniques confer only modest skull transparency due to stringent time constraints and suboptimal chemical recipes. In this work, we developed a head-mounted optically transparent skull (HOTS) window technique. The head-mounted design overcomes clearing time constraints by extending the in vivo skull-clearing process from anesthetized, head-stabilized mice to awake, freely behaving mice. The clearing cocktail was substantially improved over previous recipes via systematic chemical screening tailored to the composition and structure of the skull. Through a series of in vivo and ex vivo experiments, we demonstrated that the HOTS window technique exhibits superior clearing efficacy, good reversibility, favorable biosafety, and broad application prospects. In particular, the HOTS window technique enables transcranial two-photon imaging of neurons at an unprecedented depth, exceeding 800 mu m below the pia (permitting visualization layer 5 neurons), in adult mice, substantially outperforming existing skull-clearing methods and the use of thinned-skull windows. The achieved depth even rivals that of open-skull imaging. Even more critically, the HOTS window technique allows sensitive detection of subtle neuronal activity such as calcium transients evoked by whisker stimulation under anesthesia and faint but heterogeneous cAMP dynamics, through the skull, enabling functional investigations that were once restricted to destructive cranial windows. (c) 2025 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement

Over recent years, the LUMinescent AntiBody Sensor (LUMABS) system, utilizing bioluminescence resonance energy transfer (BRET), has emerged as a highly effective method for antibody detection. This system incorporates NanoLuc (Nluc) as the donor and fluorescent protein (FP) as the acceptor. However, the limited Stokes shift of FP poses a challenge, as it leads to significant spectral cross-talk between the excitation and emission spectra. This issue complicates the implementation of multiplexed detection. To address this challenge, we present an innovative enhancement to the LUMABS sensor with quantum dots (QDs) as the acceptor instead of FP. The use of QDs offers several advantages over those of traditional FP-based sensors. The biotin-avidin system facilitates the flexible interchangeability of QDs, allowing for a more convenient multicolor sensor construct. The new QD-LUMABS system overcomes the limitations of spectral cross-talk and provides better spectral separation. This breakthrough enables the successful implementation of multiplexed detection for multiple targets simultaneously. Results demonstrated that the wavelength-tunable QD-LUMABS sensors achieved picomolar-level detection limits for antibodies and that this sensor-construction strategy was generally applicable among various epitopes and their antibodies. Furthermore, this sensor displayed excellent duplexing capabilities. These features underscore its potential for future clinical disease diagnosis applications.

Bacterial second messengers are crucial for transmitting environmental information to cells. However, quantifying their information transmission capacity remains challenging. Here we develop a framework for quantifying information processing in cellular signalling systems. We engineer an isolated cyclic adenosine monophosphate (cAMP) signalling channel in Pseudomonas aeruginosa using targeted gene knockouts, optogenetics and a fluorescent cAMP probe. This design enables precise optical control and real-time monitoring of cAMP dynamics. By integrating experimental data with information theory, we reveal the optimal frequency for light-mediated cAMP signalling that maximizes information transmission, reaching about 40 bits per hour. This rate correlates strongly with cAMP degradation kinetics and uses a two-state encoding scheme. Our findings suggest a mechanism for fine-tuned regulation of multiple genes through temporal encoding of second-messenger signals, providing insights into bacterial adaptation strategies.

Primary cilia are cellular antennae emanating from vertebrate cell surfaces to sense and transduce extracellular signals intracellularly to regulate cell behavior and function. However, their signal sensing and physiological functions in neocortical neurons remain largely unclear. Here, we show that, in response to various animal stressors, primary cilia in the mouse prefrontal cortex (PFC) exhibit consistent axonemal elongation. Selective removal of excitatory neuron primary cilia in the prefrontal but not sensory cortex leads to a reduction in animal stress sensing and response. Treatment with corticosterone, the major stress hormone, elicits an increase in primary ciliary cyclic adenosine 3',5'-monphosphate (cAMP) level in PFC excitatory neurons and a decrease in neuronal excitability dependent on primary cilia. Suppression of primary ciliary protein kinase A (PKA) activity in PFC excitatory neurons reduces animal stress. These results suggest that excitatory neurons in the PFC are involved in sensing and regulating animal stress via primary ciliary cAMP/PKA signaling.

Soft tissue trauma can cause immune system disturbance and neuropathological invasion, resulting in heterotopic ossification (HO) due to aberrant chondrogenic differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanisms behind the interaction between the immune and nervous systems in promoting HO pathogenesis are unclear. In this study, we found that mast cell-specific deletion attenuated localized tissue inflammation, with marked inhibition of HO endochondral osteogenesis. Likewise, blockage of nerve growth factor (NGF) receptor, known as tropomyosin receptor kinase A (TrkA), led to similar attenuations in tissue inflammation and HO. Moreover, while NGF/TrkA signaling did not directly affect MSCs chondrogenic differentiation, it modulated mast cell activation in traumatic soft tissue. Mechanistically, lipid A in LPS binding to TrkA enhanced NGF-induced TrkA phosphorylation, synergistically stimulating mast cells to release neurotrophin-3 (NT3), thereby promoting MSC chondrogenic differentiation in situ. Finally, analysis of single-cell datasets and human pathological specimens confirmed the important role of mast cell-mediated neuroinflammation in HO pathogenesis. In conclusion, NGF regulates mast cells in soft tissue trauma and drives HO progression via paracrine NT3. Targeted early inhibition of mast cells holds substantial promise for treating traumatic HO.

Brainbow is a genetic cell-labeling technique that allows random colorization of multiple cells and real-time visualization of cell fate within a tissue, providing valuable insights into understanding complex biological processes. However, fluorescent proteins (FPs) in Brainbow have distinct excitation spectra with peak difference greater than 35 nm, which requires sequential imaging under multiple excitations and thus leads to long acquisition times. In addition, they are not easily used together with other fluorophores due to severe spectral bleed-through. Here, we report the development of a single-wavelength excitable Brainbow, UFObow, incorporating three newly developed blue-excitable FPs. We have demonstrated that UFObow enables not only tracking the growth dynamics of tumor cells in vivo but also mapping spatial distribution of immune cells within a sub-cubic centimeter tissue, revealing cell heterogeneity. This provides a powerful means to explore complex biology in a simultaneous imaging manner at a single-cell resolution in organs or in vivo. UFObow is a single-wavelength excitable Brainbow technique, incorporating three newly developed blue-excitable fluorescent proteins. This method facilitates mapping of immune cells' spatial distribution at a single-cell resolution.

Bacterial second messengers are crucial for transmitting environmental information to cellular responses. However, quantifying their information transmission capacity remains challenging. Here, we engineer an isolated cAMP signaling channel in Pseudomonas aeruginosa using targeted gene knockouts, optogenetics, and a fluorescent cAMP probe. This design allows precise optical control and real-time monitoring of cAMP dynamics. By integrating experimental data with information theory, we reveal an optimal frequency for light-mediated cAMP signaling that maximizes information transmission, reaching about 40 bits/h. This rate correlates strongly with cAMP degradation kinetics and employs a two-state encoding scheme. Our findings suggest a mechanism for fine-tuned regulation of multiple genes through temporal encoding of second messenger signals, providing new insights into bacterial adaptation strategies. This approach offers a framework for quantifying information processing in cellular signaling systems.

We introduce an all-optical technique that enables volumetric imaging of brain-wide calcium activity and targeted optogenetic stimulation of specific brain regions in unrestrained larval zebrafish. The system consists of three main components: a 3D tracking module, a dual-color fluorescence imaging module, and a real-time activity manipulation module. Our approach uses a sensitive genetically encoded calcium indicator in combination with a long Stokes shift red fluorescence protein as a reference channel, allowing the extraction of Ca2+ activity from signals contaminated by motion artifacts. The method also incorporates rapid 3D image reconstruction and registration, facilitating real-time selective optogenetic stimulation of different regions of the brain. By demonstrating that selective light activation of the midbrain regions in larval zebrafish could reliably trigger biased turning behavior and changes of brain-wide neural activity, we present a valuable tool for investigating the causal relationship between distributed neural circuit dynamics and naturalistic behavior.

The coincidence between conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning; however, the mechanism regulating the duration of this temporal window remains unclear. Here, we found that serotonin (5-HT) bi-directionally regulates the coincidence time window of olfactory learning in Drosophila and affects synaptic plasticity of Kenyon cells (KCs) in the mushroom body (MB). Utilizing GPCR-activation-based (GRAB) neurotransmitter sensors, we found that KC-released acetylcholine (ACh) activates a serotonergic dorsal paired medial (DPM) neuron, which in turn provides inhibitory feedback to KCs. Physiological stimuli induce spatially heterogeneous 5-HT signals, which proportionally gate the intrinsic coincidence time windows of different MB compartments. Artificially reducing or increasing the DPM neuron-released 5-HT shortens or prolongs the coincidence window, respectively. In a sequential trace conditioning paradigm, this serotonergic neuromodulation helps to bridge the CS-US temporal gap. Altogether, we report a model circuitry for perceiving the temporal coincidence and determining the causal relationship between environmental events.

Facile and scalable approaches for preparing fluorescent microspheres are indispensable tools in material science due to their immense potential for reducing the tedious conditional trial-and-error experiments for every single material. In this study, we propose a microfluidic method based on oscillating electric field (OEF)-induced electrojetting to fabricate size-controllable fluorescent microspheres embedded with various fluorescent sub-stances. Briefly, an oscillating electric field is used to manipulate the size and productivity of highly uniform droplets; the size of droplets can be adjusted by simply varying the intensity and frequency of the electric field over a range of near three orders of magnitude. Subsequently, ultraviolet (UV) irradiation is used to solidify the droplets to fabricate fluorescent microspheres. To demonstrate the universality of the proposed method in different types of fluorescent substances, we use fluorescein sodium (FS), green fluorescent protein (GFP), and CdTe quantum dots (QDs) to synthesize fluorescent microspheres. The prepared microspheres show narrow size distribution (average coefficient of variation below 2.1%) and intense fluorescence emission. Overall, the pro-posed method is a novel and facile approach for synthesizing fluorescent microspheres, showing great promise for the preparation of polymer, functionalized and other materials with potential applications in various fields of biomaterials and biochemical assays.

Genetically encoded voltage indicators (GEVIs) allow the direct visualization of cellular membrane potential at the millisecond time scale. Among these, red-emitting GEVIs have been reported to support multichannel recordings and manipulation of cellular activities with reduced autofluorescence background. However, the limited sensitivity and dimness of existing red GEVIs have restricted their applications in neuroscience. Here, we report a pair of red-shifted opsin-based GEVIs, Cepheid1b and Cepheid1s, with improved dynamic range, brightness, and photostability. The improved dynamic range is achieved by a rational design to raise the electrochromic Förster resonance energy transfer efficiency, and the higher brightness and photostability are approached with separately engineered red fluorescent proteins. With Cepheid1 indicators, we recorded complex firings and subthreshold activities of neurons on acute brain slices and observed heterogeneity in the voltage‑calcium coupling on pancreatic islets. Overall, Cepheid1 indicators provide a strong tool to investigate excitable cells in various sophisticated biological systems.

Bright monomeric near-infrared fluorescent proteins (NIR-FPs) are useful as markers for labeling proteins and cells and as sensors for reporting molecular activities in living cells and organisms. However, current monomeric NIR-FPs are dim under excitation with common 633/635/640 nm lasers, limiting their broad use in cellular/subcellular level imaging. Here, we report a bright monomeric NIR-FP with maximum excitation at 633 nm, named mIFP663, engineered from Xanthomonas campestris pv Campestris phytochrome (XccBphP). mIFP663 has high molecular brightness with a large extinction coefficient (86,600 M-1 cm-1) and a decent quantum yield (19.4%), and high cellular brightness that is 3-6 times greater than those of spectrally similar NIR-FPs in HEK293T cells in the presence of exogenous BV. Moreover, we demonstrate that mIFP663 is able to label critical cellular and viral proteins without perturbing subcellular localization and virus replication, respectively. Finally, with mIFP663, we engineer improved bimolecular fluorescence complementation (BiFC) and new bioluminescent resonance energy transfer (BRET) systems to detect protein-protein interactions in living cells.

cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum Δ F / F 0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a K d of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

Red fluorescent proteins are useful as morphological markers in neurons, often complementing green fluorescent protein-based probes of neuronal activity. However, commonly used red fluorescent proteins show aggregation and toxicity in neurons or are dim. We report the engineering of a bright red fluorescent protein, Crimson, that enables long-term morphological labeling of neurons without aggregation or toxicity. Crimson is similar to mCherry and mKate2 in fluorescence spectra but is 100 and 28% greater in molecular brightness, respectively. We used a membrane-localized Crimson-CAAX to label thin neurites, dendritic spines and filopodia, enhancing detection of these small structures compared to cytosolic markers.

SignificancePhotoacoustic (PA) imaging can provide information about deep-seated biological tissues. Unfortunately, signal intensity and resolution can be limited by background signals arising from endogenous chromophores. Photochromism, the use of agents that go from colorless to colored, has been suggested to be a promising avenue to overcome this limitation. However, a viable method that allows practical applications of this so-called background-suppressed PA imaging is still lacking. Here, we report the engineering of a phytochrome-based reporter protein (mDrBphP-PCMm/F469W) that displays high photoswitching contrast. Its expression inEscherichia colienables point-specific imaging within the tumor region over several days and permits PA monitoring of deep-seated tumor tissues via reduction of background signals.

With the development of fluorescent proteins (FPs) and advanced optical microscopy techniques, Förster or fluorescence resonance energy transfer (FRET) has become a powerful tool for real-time noninvasive visualization of a variety of biological processes, including kinase activities, with high spatiotemporal resolution in living cells and organisms. FRET can be detected in appropriately configured microscopes as changes in fluorescence intensity, lifetime, and anisotropy. Here, we describe the preparation of samples expressing FP-based FRET sensors for RhoA kinase, intensity- and lifetime-based FRET imaging, and postimaging data analysis.