Fluorescence lifetime imaging of compartmental pH dynamics using red fluorescent protein sensors in live cells

Abstract

pH regulation plays a crucial role in protein function, cell metabolism, intracellular degradation, and a wide range of other physiological processes. Monitoring perturbations in local pH environments due to cellular activities or diseases, such as increased neuron activity, receptor trafficking, or extracellular acidosis in a tumor microenvironment, would better our understanding of disease pathology. Live‐cell imaging with fluorescent protein‐based sensors allows us to monitor broad pH changes; however, there are currently limited options to quantitatively measure pH dynamics with subcellular resolution in live specimens. Concentration and intensity dependence of the fluorophore, sample thickness, and photo‐bleaching are limiting factors, and additionally, most of the current genetically encoded pH sensors are generated from green fluorescent protein, limiting our ability to perform multiplex imaging to monitor pH simultaneously in different compartments. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. Utilizing this property, we can monitor discrete compartmental changes in pH using fluorescence lifetime imaging microscopy (FLIM), while opening up a wider selection of potential fluorescent sensors for multi‐channel imaging. It is the goal of this project to develop genetically‐encoded red fluorescent pH sensors to quantitatively measure micro‐environmental pH changes using fluorescence lifetime. Here we present in vitro and live‐cell characterization; in proof‐of‐concept studies we have utilized FLIM to measure the pH of different subcellular compartments simultaneously in real‐time. Our results demonstrate our progress towards understanding the nuances of subcellular pH and aberrant cell behavior, and how the role of pH is affected in both healthy and disease states.Support or Funding InformationEmily Haynes is supported by her Charles H. Viol Chemistry Fellowship. We also gratefully acknowledge support from the Showalter Fund and from National Institutes of Health grants R21 NS092010 and R21 EY026425.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fluorescent naphthalimide boronates as theranostics: structural investigations, confocal fluorescence and multiphoton fluorescence lifetime imaging microscopy in living cells

“…It has been shown that the magnitude of the emission lifetime may changes when a fluorescent probe binds to a target that could affect its environment. 45 …”

“…Excited state lifetime measurements evaluate an absolute length scale (on the nanometre range), 37 and, as such, imaging assays using FLIM techniques are less vulnerable to factors such scattered light, photobleaching, nonuniform illumination of the probe, light path length, or laser intensity variations as these can normally be 'gated' out or easily corrected for. Therefore, 2P FLIM has been widely used in imaging cellular protein interactions [38][39][40] and conformational changes, 41 viscosity, 42,43 temperature, 44 pH, 45 ions 46 and oxygen concentrations. 47,48 FLIM generated images based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample on a pixel-by-pixel basis, whereby very high resolution at the diffraction limited level can be obtained.…”

“…It has been shown that the magnitude of the emission lifetime may changes when a fluorescent probe binds to a target that could affect its environment. 45 To further elucidate the cellular uptake and biodistribution within cells, compounds 1-BPin and 2-BPin and the corresponding b-D-glucan hybrids of compounds 1-2 were investigated by two photon fluorescence lifetime imaging correlated with single photon confocal imaging, and results are depicted in Fig. 8, 9 and Table 1.…”