Calcium-sensitive photoacoustic probe for noninvasive extracellular calcium monitoring

Changes in extracellular calcium concentrations ([Ca²⁺]e) can mediate a variety of biological responses in both excitable and nonexcitable cells. These changes can be seen in both physiological and pathological conditions; however, little is still known about their effects to neuronal excitability. Fluorescent calcium probes are essential tools for studying the fluctuation of calcium ions both in and out of cells. Unfortunately, current techniques utilizing these calcium probes have many limitations that have yet to be addressed, including lack of penetration depth and concurrent multiple site analysis in the whole brain. For example, fluorescence imaging suffers from light diffusion, a fundamental constraint that limits the imaging depth in tissue (< 1 mm). Photoacoustic tomography (PAT) has emerged as a promising imaging modality that overcomes this challenge. In this paper, we utilized a near infrared (NIR) ratiometric calcium fluorescent probe (Ca-NIR) as a unique photoacoustic calcium probe. Ca-NIR is based on fusing a selective calcium ligand BAPTA (1,2-bis-(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid) moiety to a dihydroxanthenehemicyanine fluorophore. We report the use of Ca-NIR as an efficient PA generating agent in various artificial cerebral-spinal fluid (aCSF) solutions with varying Ca²⁺ concentrations. Our result indicates high sensitivity of Ca-NIR to [Ca²⁺]_e fluctuations in aCSF and great potential of utilizing Ca-NIR in PAT as a method for noninvasive whole brain [Ca²⁺]_e imaging.