Noninvasive, deep-tissue imaging techniques are critical for advancing our understanding of neural circuits across various areas of neuroscience. Among these, near-infrared (NIR) wavelengths have emerged as promising tools, offering deeper tissue penetration due to reduced light absorption and scattering, while minimizing tissue damage. However, the limited brightness of NIR fluorescent proteins remains a major challenge for their effective application in in vivo imaging. 

This study addresses this challenge by focusing on optimizing the brightness of NIR fluorescent proteins, while preserving their essential spectral properties. A key obstacle, the limited binding affinity of biliverdin (cofactor with NIR fluorescent proteins) in mammalian tissues, has further complicated the development of NIR sensors. To overcome this, we performed a quantitative analysis of several novel NIR fluorescent proteins, evaluating their fluorescence intensity and spectral characteristics.

Through this analysis, we identified several top-performing candidates that demonstrated significant potential to enhance noninvasive deep-tissue imaging. These findings highlight the promise of NIR fluorescent proteins, especially when paired with selective labeling strategies, such as cell-type-specific promoters or organelle-targeting sequences. The improved brightness of these proteins paves the way for further refinement, potentially leading to more precise and efficient methods for deep-tissue imaging and related neurological research and beyond.