Fluorescent indicators allow the monitoring of physiological parameters in biological tissues by measuring changes in the brightness of the indicator upon binding of its ligand. Quantitative measurement of these parameters in vivo with high spatio-temporal resolution using multiphoton microscopy often requires the use of fluorescence ratiometric measurements. However, ratiometric measurements can be biased at depth in biological tissues due to absorption and scattering of the photons involved in the process.
Here we have developed a mathematical model that takes into account the chemical properties of the sensors, the spectral optical properties of the biological tissue and the optical system to provide quantitative measurements of the concentration of the sensor ligand. We have also developed a software implementing this model. Both can be used to obtain unbiased measurements of different physiological biomarkers from multiphoton images.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.