OCaMP: A New Calcium Indicator for Neural Imaging

This post was written by Abhi Aggarwal, from University of Calgary.

Over the past few decades, genetically encoded calcium indicators (GECIs) have become a vital tool in neuroscience research. These fluorescent proteins light up in response to calcium, which is more than just the stuff that makes bones strong; it plays critical roles in nerve and muscle function, hormone release, and cell signaling throughout the body. Because calcium levels change rapidly during neuronal activity, scientists can use GECIs to watch neurons fire under the microscope. To add another tool to our toolbox, our team recently developed a new calcium indicator, OCaMP.

The challenge: Expanding the fluorescent toolbox

Modern neuroscience experiments often rely on multiplexing, where multiple fluorescent tools are used simultaneously to monitor or control different cell types or signals in the brain. For this to work, each indicator needs to emit a different color of light and be compatible with the lasers available in your microscope setup.

Figure 1: Excitation (dotted) and emission (solid) spectra for various teal, green, yellow, orange, and red fluorophores. The spectra for mOrange2 (scaffold of OCaMP) are shaded in orange. Data from FPbase.org.

Scientists have developed calcium indicators spanning nearly the entire visible spectrum (Figure 1). The GCaMP series (e.g., jGCaMP8) remains the gold standard for green fluorescence, while red indicators jRGECO1a and jRCaMP1a are widely used as well. To complement these tools, we recently developed a new orange calcium indicator, OCaMP (Aggarwal et al., 2025), based on the fluorescent protein mOrange2 (Figure 2).

Figure 2: OCaMP structure and mechanism of action. Reproduced from Aggarwal et al. 2025 under a CC-BY-NC-ND 4.0 International license.

Why orange?

OCaMP’s emission peak at 565 nm fits neatly between yellow and red fluorophores, allowing it to be paired with green, yellow, and/or red sensors in multicolor experiments. With OCaMP, researchers can image multiple brain regions or cell types at once using existing indicators. For example, you might want to use OCaMP to monitor Ca²⁺ signals, while using  iGluSnFR4 to monitor glutamate in green/yellow and RdLight to monitor dopamine in red. 

However, current red and green GECIs aren’t ideal for two-photon imaging with newer lasers in the range of 1000–1080 nm. These industrial fiber lasers are becoming increasingly common due to their low cost and high power, but because they are non-tunable, their usefulness depends on selecting a fluorophore matched to their excitation wavelength. OCaMP is well-suited for these lasers, with two-photon excitation at 1030 nm, enabling two-photon imaging without compromising on brightness, ∆F/F₀, or signal-to-noise ratio (Figure 3).

Figure 3: One- and two-photon excitation and emission spectra of OCaMP. 1P excitation peak is 545 nm, 1P emission peak is 565 nm, and 2P excitation peak is 1050 nm. Reproduced from Aggarwal et al. 2025 under a CC-BY-NC-ND 4.0 International license.

Better performance = better data

In side-by-side tests with popular red and yellow GECIs, OCaMP shows:

• Higher ΔF/F₀ (the measure of signal change during calcium events),
• Greater brightness,
• Less signal distortion from photoswitching or photobleaching
• Improved signal-to-noise ratio (SNR).

Figure 4: Calcium titration curves for OCaMP and various red and yellow GECIs measured with soluble protein. Reproduced from Aggarwal et al. 2025 under a CC-BY-NC-ND 4.0 International license.

Figure 5: A) Simultaneous fluorescence (upper trace) and electrophysiological recordings (lower trace) using OCaMP expressed in mouse cortex. Bottom: Zoomed-in view of traces corresponding to the dashed box above. The number of action potential (AP) spiking events are indicated below the trace; asterisk indicates a single AP. B) Averaged fluorescence responses to increasing numbers of evoked APs in neurons expressing OCaMP or jRGECO1a. Reproduced from Aggarwal et al. 2025 under a CC-BY-NC-ND 4.0 International license.

Using OCaMP in your lab

Several OCaMP constructs are now available through Addgene. Whether you're working with cell cultures, slice preparations, or in vivo imaging, there's a version that fits your setup:

Plasmid	Backbone	Promoter
pRSET OCaMP	pRSET	T7
pAAV-hSYN1-NES-OCaMP-WPRE	pAAV	hSyn
pAAV-EF1a-FLEX(FLP)-OCaMP-WPRE	pAAV	Ef1a
pAAV-hSyn-FLEX(cre)-OCaMP-WPRE	pAAV	hSyn
pAAV-CMV-OCaMP-WPRE	pAAV	CMV

The Lohman lab has a limited quantity of AAVs that can be requested by contacting Alexander Lohman (alex.lohman@ucalgary.ca). 

For a deeper dive into the science behind OCaMP, including detailed characterization as protein or in neuronal cultures, zebrafish, and mouse brain, check out the preprint on bioRxiv.

Find OCaMP plasmids available at Addgene!

Abhi Aggarwal is a medical student at the University of Calgary and a research associate with Alexander Lohman at the Hotchkiss Brain Institute. He previously worked at The Allen Institute for Neural Dynamics and HHMI’s Janelia Research Campus. His research focuses on developing biosensors and tools for neural imaging. Follow him on Bluesky (@abhiaggarwal.bsky.social‬).

 

References and Resources

References

Aggarwal, A, et al. (2025). A sensitive orange fluorescent calcium ion indicator for imaging neural activity. bioRxiv 2025.07.28.667269; doi: https://doi.org/10.1101/2025.07.28.667269.

Additional Resources on the Addgene Blog

• Viral Vectors 101: Calcium Sensors
• New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor
• A Practical Approach to Choosing the B(right)est Fluorescent Protein

Additional Resources on addgene.org

• Fluorescent Proteins: Biosensor Plasmids
• In-Stock AAV Vectors: Biosensors
• AAV Packaged on Request