Nature Methods | PinkyCaMP: The Brightest, Non-Photoswitchable Red Calcium Indicator Yet!

PinkyCaMP: an mScarlet-based calcium sensor with enhanced brightness, photostability and multiplexing capabilities | Nature Methods

1. Research Background and Challenges

• Low brightness and poor signal-to-noise ratio 
• Blue-light-induced photoswitching, causing reversible fluorescence changes that interfere with optogenetic experiments
• Lysosomal aggregation and poor photostability 
• Limited tissue penetration and multicolor multiplexing capabilities

2. Design and Construction of PinkyCaMP

Figure 1 | Development and characterization of PinkyCaMP
 

3. Key Physicochemical and Optical Properties (Critical Data)

4. Multi-Level Functional Validation

(1). In Vitro Cellular Level

Figure 2 | Basic characterization of PinkyCaMP in cultured cells

Figure 3 | Characterization of PinkyCaMP in cultured neurons
 

(2). Ex Vivo Brain Slice Level

Cortical organotypic slices: Baseline brightness and absolute signal intensity surpass all red GECIs, comparable to GCaMP6f.

Figure 4 | Comparison of PinkyCaMP with other GECIs in cortical organotypic slices

Acute hippocampal slices: Brightness 2× that of RCaMP3, with SNR = 63.5 ± 7.9 (RCaMP3: 33.7 ± 4.8).

Figure 5 | Comparison of PinkyCaMP with other GECIs in acute brain slices
 

(3). In Vivo Animal Level

Fiber photometry: Calcium signals in the prefrontal cortex and dentate gyrus are clear, compatible with blue-light optogenetic control without interference.

Figure 6 | Integration of PinkyCaMP with blue-light-sensitive optogenetic techniques

Two-photon imaging: Excitation at 1040 nm allows recording of CA1 hippocampal neurons in awake, head-fixed mice.

Figure 7 | Two-photon imaging of PinkyCaMP in freely moving mice
 

Miniature microscopy: Single- and two-photon imaging in freely moving mice is stable and reliable.

Figure 8 | Compatibility of PinkyCaMP with miniature microscope imaging systems

 

(4). Multicolor Multiplexing Capability

🔹Combined with GCaMP8s: Excitatory and inhibitory neurons can be labeled separately with minimal crosstalk.

🔹Combined with sDarken (serotonin sensor): Allows simultaneous recording of Ca²⁺ and serotonin.

🔹Combined with blue-light opsins (stCoChR/stGtACR2): Enables all-optical manipulation and recording without artifacts.

5. Advantages and Limitations

(1). Key Advantages

🔹One of the brightest red GECIs in the world, with brightness approaching that of the GCaMP series

🔹Completely free of blue-light photoswitching, compatible with all-optical experiments

🔹Excellent photostability, minimal lysosomal aggregation

🔹Deep tissue penetration with low phototoxicity 

(2). Main Limitations

🔹Slower kinetics (half-rise time ~670 ms; half-decay time ~5.6 s)

🔹Not suitable for precise high-frequency action potential resolution 

 

Brain Case Biotech also offers custom services.
For more information, please contact
bd@ebraincase.com