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Powerful Research Tools | In-Depth Analysis of 8 Red Calcium Sensors to Unlock Neural Activity Mysteries!

Release time：2025-05-29 16:24:09
Calcium signals recording is a technique that uses calcium indicators to detect the concentration of calcium ions within tissues. This technology is widely applied in the fields of neuroscience, cell biology, physiology, developmental biology, and pharmacology. In neuroscience, it is mainly used to study the signal emissions triggered by changes in the ion concentration of nerve cells. In physiology, it primarily focuses on calcium signaling in muscle movement, particularly in cardiac cells. In cell biology, it is mainly used to study signal transduction and ion channels. In developmental biology, it is used to investigate mechanisms of egg fertilization, while in pharmacology, it is mainly applied in drug screening and pharmacodynamics.

As an important tool, red calcium sensors are gradually becoming an indispensable assistant for researchers. Today, we will provide a detailed introduction to several red calcium sensors to help you better understand their design principles and characteristics.
 


01. jRCaMP1 Series: 560 nm excitation/580 nm emission

✨Design Principle

Developed based on mRuby, fused with M13 peptide interacting with calmodulin (CaM) and its binding partners. Through structure-guided mutagenesis and neuronal screening between RFP and CaM, CaM and M13, as well as within CaM itself, the sensitivity for detecting neural activity was enhanced.

✨Characteristics

High Sensitivity: jRCaMP1a and jRCaMP1b show a 24-fold and 13-fold increase in sensitivity to detect a single action potential (AP) stimulus compared to their parent indicators.
Spectral Properties: Similar absorption and emission spectra to the parent constructs.
Photostability: Like jRGECO1a, jRCaMP1a and jRCaMP1b do not exhibit photobleaching under blue light exposure, making them suitable for use in combination with optogenetics.
 

02. jRGECO1a: 560 nm excitation/580 nm emission

✨Design Principle

Based on mApple, it is fused with CaM and M13 peptide. Through mutagenesis between RFP and CaM, CaM and M13, as well as within CaM itself, the performance of the indicator was enhanced.

✨Characteristics

High Sensitivity: It is the most sensitive indicator, with a ΔF/F₀ amplitude for a single AP stimulus 8.5 times greater than R-GECO1, and a faster rise time.
Spectral Properties: Similar to other mApple-based indicators, it exhibits photoconversion properties under blue light exposure.
Comparison with Other Indicators: It has similar performance to GCaMP6 indicators and can be used for detecting neural activity.
 

03. NIR - GECO2 and NIR - GECO2G: 640 nm excitation/685 nm emission

✨Design Principle

Origin: Both variants originated from NIR-GECO1. By genetically modifying NIR-GECO1, including random mutagenesis and screening steps, these two second-generation variants were developed. The second-generation NIR-GECO2 was constructed by replacing the mIFP portion with the brighter homolog miRFP.
Specific Mutations: NIR-GECO2 introduced mutations such as T234I, S251T, E259G, Q402E, F463Y, and T478A. NIR-GECO2G further introduced T251S and S347G mutations on the basis of NIR-GECO2.

✨Characteristics

Increased Sensitivity: NIR-GECO2G shows a significant improvement in sensitivity for detecting calcium ion changes associated with neural activity compared to NIR-GECO1. For example, its ΔF/F₀ response to a single AP stimulus is approximately 3.7 times higher than NIR-GECO1.
Spectral Properties: It shares similar fluorescence spectral properties, peak maximum, extinction coefficient, quantum yield, and pKa with NIR-GECO1.
Other Characteristics: It has a high calcium affinity and performs well both in vitro and in vivo, accurately reporting changes in calcium ion concentrations.
 

04. iGECI: 630 nm excitation/670 nm emission

✨Design Principle

iGECI is based on the Cameleon-like GECI scaffold, combining recently described bright monomeric near-infrared fluorescent proteins, miRFP670 and miRFP720. Random mutagenesis and screening of the amino acid sequence in the calcium-sensitive module were conducted to optimize the sensor's performance, enhancing its sensitivity and specificity to calcium ions.

✨Characteristics

Near-Infrared Fluorescence: Excitation and emission wavelengths are in the near-infrared region, allowing effective tissue penetration, reducing light scattering and absorption, and enabling high-resolution imaging of deeper tissues.
High Brightness and Photostability: It exhibits high brightness and excellent photostability, maintaining stable fluorescence signals during extended imaging sessions.
High Sensitivity: It can sensitively detect changes in intracellular calcium ion concentrations, with a high sensitivity to single action potential (AP) responses.
Fast Kinetic Response: It has rapid rise and decay times, allowing for timely reflection of changes in neuronal activity.

05. HaloCaMP1a and HaloCaMP1b: 640 nm excitation/655 nm emission

✨Design Principle

Both HaloCaMP1a and HaloCaMP1b are chemical-genetic calcium ion sensors based on the HaloTag protein, where cpHaloTag is linked with CaM and the CaM-binding peptide, mimicking the design of GCaMP. Based on photoinduced electron transfer (PET), changes in protein conformation affect the balance between the colorless, non-fluorescent lactone (L) form and the colored, fluorescent zwitterionic (Z) form, thereby altering fluorescence intensity.

✨Characteristics

Spectral Properties: After binding with the JF635-HaloTag ligand, the fluorescence excitation and emission spectra of HaloCaMP1a and HaloCaMP1b exhibit a red shift. The excitation and emission maxima of HaloCaMP1a are 640nm and 653nm, respectively, while for HaloCaMP1b, they are 642nm and 655nm, placing the spectra in the far-red region.
High Sensitivity: HaloCaMP1a has a dissociation constant (Kd) of 190nM, with a ΔF/F0 of 5.0; HaloCaMP1b has a dissociation constant (Kd) of 43nM, with a ΔF/F0 of 9.2. Due to its lower Kd and higher ΔF/F0, HaloCaMP1b exhibits greater sensitivity. In neuronal culture experiments, it can effectively detect single action potentials.
 

06. WHaloCaMP1a: The excitation and emission wavelengths vary when bound to different dye ligands.

✨Design Principle

WHaloCaMP is based on HaloTag7, with the G171 mutation to tryptophan introducing a fluorescence quenching mechanism, and the insertion of CaM and related peptide segments at position R179. It primarily relies on the principle of photoinduced electron transfer (PET), where tryptophan reversibly quenches the bound dye. When calcium ions bind, the conformational change in the protein affects the PET process, which in turn alters fluorescence intensity.

✨Characteristics

Spectral Properties: The excitation spectrum has good separation from many blue-excited optogenetic tools, ensuring that it does not induce observable membrane depolarization in neurons expressing CheRiff during recording. Its near-infrared emission can be multiplexed with existing fluorescence protein-based indicators.
Fluorescence Intensity and Sensitivity: Compared to other calcium indicators, WHaloCaMP exhibits a significant increase in fluorescence intensity. For instance, when bound with near-infrared dye ligands, the fluorescence intensity increases by 7 times, making it more than twice as bright as jGCaMP8s and 40 times brighter than iGECI. It shows excellent performance in neuronal culture experiments and can detect single action potentials.
Versatile Functionality: WHaloCaMP can be used not only for detecting neuronal activity but also for emitting multiple colors of fluorescence through binding with different dye ligands, making it suitable for various imaging needs. Additionally, it can be used as a fluorescence lifetime imaging microscope (FLIM) sensor for quantitative measurement of intracellular calcium ion concentrations.
 

07. RCaMP3: 560 nm excitation/610 nm emission

✨Design Principle

RCaMP3 is developed based on R-GECO1. A series of mutations were introduced to blue-shift the excitation spectrum and generate a larger dynamic range. Additionally, a self-cleaving peptide (F2A) was used to replace the nuclear output signal of jRGECO1a.

✨Characteristics

Spectral Properties: RCaMP3 has a more blue-shifted excitation spectrum, making it suitable for two-photon imaging (1040 nm).
Sensitivity and Dynamic Range: It demonstrates high sensitivity and a large dynamic range in both in vitro and in vivo experiments, enabling the detection of single neuron activity.
Comparison with Other Indicators: In some experiments, RCaMP3 outperforms jRGECO1a. For instance, in response to a single action potential, RCaMP3 exhibits a higher ΔF/F₀ value
 

08. FRCaMPi and SomaFRCaMPi: 566 nm excitation/594 nm emission

✨Design Principle

FRCaMPi Design: The design of FRCaMPi improves the indicator's calcium ion binding affinity by altering the terminal connection mode of FRCaMP through topological inversion.
SomaFRCaMPi Design: The C-terminal of FRCaMPi is connected with ribosomal protein L10 (RPL10), allowing it to specifically localize to the neuronal soma, thereby reducing glial contamination and improving signal accuracy.

✨Characteristics

High Sensitivity: FRCaMPi and SomaFRCaMPi exhibit high sensitivity in neurons, capable of detecting subtle calcium ion changes.
Strong Specificity.
Soma Localization: SomaFRCaMPi specifically localizes the signal to the neuronal soma, reducing glial signal interference and improving signal quality.
Reduced Correlation: In experiments with mice and zebrafish, SomaFRCaMPi reduces erroneous correlations between neuronal activities, enhancing imaging accuracy.
Other Properties: Both in vitro and in vivo experiments show excellent stability and photostability, making it suitable for long-duration imaging.
 

Application Scenarios

1. Neuron Activity Detection

In Vivo Recording: These red calcium sensors can be used for in vivo detection of neuronal activity. For example, by expressing these indicators in animal models such as mice, fruit flies, zebrafish, and Caenorhabditis elegans, real-time monitoring of neuronal activity changes can be achieved.
Cell Culture: In cultured neurons, these indicators can respond to changes in intracellular calcium ion concentrations, thereby reflecting the activity state of the neurons.

2. Neural Circuit Research

Multi-Neuron Recording: By expressing red calcium sensors in different neurons, multiple neurons' activities can be monitored simultaneously to study the functionality and connectivity of neural circuits.
Activity Correlation Analysis: For example, by analyzing the calcium ion signal correlations between different neurons, the interactions and information transfer modes between neurons in the neural circuit can be revealed.

3. Behavioral Research

Motor Behavior: In studying animal motor behavior, these indicators can record neuronal activity related to movement, helping to understand the neural mechanisms of motor control.
Sensory Stimulus Response: In visual stimulus experiments, red calcium sensors can detect the response of visual cortex neurons to visual stimuli, revealing the neural processes involved in visual information processing.

4. Drug Screening and Neurobiology Research

Drug Mechanism of Action Study: By expressing red calcium sensors in cell or animal models and administering different drug treatments, the effects of drugs on neuronal activity can be studied, revealing the mechanisms of drug action.

Summary

These red calcium sensor products have unique features in terms of design principles, characteristics, excitation wavelengths, and application scenarios. Their development provides essential tools for neuroscience research, aiding in the deeper understanding of the mechanisms of neuronal activity and the functionality of neural circuits.
 
References
1、Dana H, Mohar B, Sun Y, et al. Sensitive red protein calcium indicators for imaging neural activity. Elife. 2016;5:e12727. 
2、Qian Y, Cosio DMO, Piatkevich KD, et al. Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging. PLoS Biol. 2020;18(11):e3000965.
3、Shemetov AA, Monakhov MV, Zhang Q, et al. A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat Biotechnol. 2021;39(3):368-377.
4、Deo C, Abdelfattah AS, Bhargava HK, et al. The HaloTag as a general scaffold for far-red tunable chemigenetic indicators. Nat Chem Biol. 2021;17(6):718-723. 
5、Farrants H, Shuai Y, Lemon WC, et al. A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging. Nat Methods. 2024;21(10):1916-1925.
6、Yokoyama T, Manita S, Uwamori H, et al. A multicolor suite for deciphering population coding of calcium and cAMP in vivo. Nat Methods. 2024;21(5):897-907.
7、A Sensitive Soma-localized Red Fluorescent Calcium Indicator for Multi-Modality Imaging of Neuronal Populations In Vivo. bioRxiv 2025.01.31.635851; doi: https://doi.org/10.1101/2025.01.31.635851.
 

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