Novel Approach for Lifetime-Proportional Luminescence Imaging Using Frame Straddling

Abstract

Optode-based chemical imaging is a rapidly evolving field that has substantially enhanced our understanding of the role of microenvironments and chemical gradients in biogeochemistry, microbial ecology, and biomedical sciences. Progress in sensor chemistry has resulted in a broadened spectrum of analytes, alongside enhancements in sensor performance (e.g., sensitivity, brightness, and photostability). However, existing imaging techniques are often costly, challenging to implement, and limited in their recording speed. Here we use the “frame-straddling” technique, originally developed for particle image velocimetry for imaging the O₂-dependent, integrated luminescence decay of optical O₂ sensor materials. The method synchronizes short excitation pulses and camera exposures to capture two frames at varying brightness, where the first excitation pulse occurs at the end of the exposure of the first frame and the second excitation pulse at the beginning of the second frame. Here the first frame truncates the luminescence decay, whereas the second frame fully captures it. The difference between the frames quantifies the integral of the luminescence decay curve, which is proportional to the luminescence lifetime, at time scales below one millisecond. Short excitation pulses avoid depopulation of the ground state of luminophores, resulting in a linear Stern–Volmer response with increasing concentrations of the quencher (O₂), which can be predicted through a simple model. This methodology is compatible with a wide range of camera systems, making it a versatile tool for various optode based chemical imaging applications. We showcase the utility of frame straddling in measuring O₂ dynamics around algae and by observing O₂ scavenging sodium dithionite particles sinking through oxygenated water.