Genetically encoded calcium indicators (GECIs) are crucial tools for real-time monitoring of intracellular calcium dynamics and cellular activities. The signal-to-baseline ratio (SBR, ΔF/F0) is a key performance parameter for GECIs, representing the change in fluorescence relative to baseline fluorescence. While efforts have focused on improving kinetic speed, increasing the maximal fluorescence change has lagged. Current GECIs, including the GCaMP6 series and NCaMP7, while bright, still face limitations in their dynamic range (DR), which restricts their ability to resolve subtle calcium changes. This study aimed to develop substantially improved GECIs with enhanced dynamic range and speed, built upon the bright mNeonGreen (mNG) fluorescent protein.

Existing GECIs utilize various strategies to achieve calcium sensing. Single-FP based indicators often employ a calcium-sensing module, such as calmodulin (CaM) and its target peptide (e.g., RS20 or M13), incorporated within a fluorescent protein. Two main design approaches have been used: a GCaMP-like design, attaching the CaM-M13 module to the termini of the fluorescent protein, and an NCaMP7-like design, inserting the module into the middle of the protein. Linker modifications and alterations to the interaction interfaces between CaM, M13, and the fluorescent protein have been shown to enhance GECI performance. However, further improvements in dynamic range have been hampered by the inherent brightness limitations of the fluorescent proteins, even those using the brightest monomeric green fluorescent proteins like mNG. This research builds upon the strengths of both the GCaMP and NCaMP7 designs to overcome these limitations.

The researchers generated a series of mNG-based GECIs (NEMO) by introducing amino acid substitutions into NCaMP7. They screened these variants in HEK293 cells, evaluating their basal fluorescence (F0), dynamic range (DR), and kinetics. Dynamic range was determined by measuring the maximal (Fmax) and minimal (Fmin) fluorescence after depleting ER Ca2+ stores with ionomycin and thapsigargin and then inducing Ca2+ entry via SOCE. Five of the best-performing constructs (NEMOs, NEMOb, NEMOm, NEMOc, and NEMOf) were chosen for further characterization. *In vitro* and *in cellulo* characterization included measuring dose-response curves, basal fluorescence (normalized against mKate using a P2A-based bicistronic vector), and photostability. Spectral properties, including excitation, emission, and absorption spectra, were also analyzed to understand the mechanism behind the improved DR. Ratiometric measurements using 405-nm and 488-nm excitation were performed, and photochromic effects were explored for absolute Ca2+ concentration quantification using the intermittent photochromism-enabled absolute quantification (iPEAQ) method. The functionality of NEMO sensors was further evaluated in various contexts: Ca2+ oscillations in HEK293 cells (induced by carbachol), weak Ca2+ signals (TG-induced Ca2+ release and SOCE, BmGr-9 mediated Ca2+ influx), and responses to graded Ca2+ influx (using Opto-CRAC). In addition to mammalian cells, the application of NEMOs was tested in *Arabidopsis thaliana* leaves. *In vivo* performance was evaluated in rat hippocampal neurons using electrical field stimulation and in mouse brain using two-photon microscopy and fiber photometry to monitor visual cortex responses to drifting gratings and striatal responses to tail-pinching stimuli, respectively.

The study successfully engineered a suite of GECIs, NEMO, with superior performance compared to state-of-the-art GECIs like GCaMP6 and NCaMP7. Key findings include: 1. **Enhanced Dynamic Range:** NEMOs displayed dynamic ranges exceeding 100-fold, significantly larger than GCaMP6m or NCaMP7 (at least 4.5 times higher). 2. **Increased Sensitivity:** NEMOs exhibited much higher signal-to-baseline ratios (SBRs) than GCaMP6s and GCaMP6f in both cultured neurons and *in vivo* experiments (e.g., peak SBR in neurons was approximately twice as high). NEMO could detect single action potentials with peak SBRs two times higher and median peak SBRs four times larger in vivo than GCaMP6s. 3. **Fast Kinetics:** NEMOf exhibited kinetics comparable to or faster than existing fast GECIs, capable of resolving neuronal responses at frequencies up to 5Hz. 4. **Ratiometric and Photochromic Capabilities:** NEMOs function as ratiometric sensors, enabling direct measurement of absolute Ca2+ concentrations. They also demonstrate photochromic properties, allowing for the use of the iPEAQ method to precisely quantify calcium levels. 5. **Improved Photostability:** NEMOs showed significantly better photostability compared to GCaMP6m, capable of withstanding substantially stronger illumination. 6. ***In vivo* Performance:** Two-photon imaging in the mouse visual cortex and fiber photometry in the striatum confirmed NEMOs superior performance in detecting calcium signals *in vivo*, exhibiting significantly higher median peak responses than GCaMP6f. 7. **Plant Applications:** NEMOm successfully detected Ca2+ oscillations near plasmodesmata in *Arabidopsis thaliana* leaves, demonstrating the applicability of the indicators to plant systems.

The development of NEMO GECIs addresses the limitations of existing GECIs by achieving a significant improvement in both dynamic range and speed without sacrificing sensitivity. The large dynamic range of NEMOs is particularly advantageous for resolving subtle calcium signals, which are often missed by current indicators. This allows for better quantification of calcium signals and enhanced resolution in both *in vitro* and *in vivo* experiments. The ratiometric and photochromic capabilities of NEMOs further expand their versatility, enabling direct measurements of absolute calcium concentration and facilitating more precise quantification of cellular activity. The *in vivo* results, obtained using both two-photon microscopy and fiber photometry, demonstrate the effectiveness of NEMOs in monitoring neuronal activity in freely behaving animals. The findings have broad implications for neuroscience research and the study of calcium-dependent processes in various organisms.

This study introduces the NEMO family of GECIs, offering a significant advance in calcium imaging technology. NEMOs combine fast kinetics, high sensitivity, a large dynamic range, and ratiometric/photochromic capabilities. Their superior performance in both *in vitro* and *in vivo* experiments establishes them as a powerful tool for studying calcium signaling in various biological systems, paving the way for more precise and detailed investigations of neuronal activity and other calcium-regulated processes. Future research could focus on expanding the spectral palette of the NEMO sensors, optimizing their expression in specific cell types, and further exploring their applications in other biological systems.

While NEMOs demonstrate significant advantages, some limitations exist. The high dynamic range necessitates careful background correction to prevent overestimation of DR values. The optimal excitation wavelength for NEMOs (980 nm) is different from that of GCaMP (920 nm), which may influence direct comparison between the two. The in vivo experiments primarily focused on neuronal activity in the visual cortex and striatum; further studies are needed to validate NEMOs performance in other brain regions and cellular contexts.