Imaging of Cellular Oxygen and Analysis of Metabolic Responses of Mammalian Cells

Abstract

Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca(2+), H(+)), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite - cellular oxygen - is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O(2) in live respiring cells and metabolic responses to cell stimulation. The techniques include conventional wide-field fluorescence microscopy to monitor relative changes in cell respiration, microsecond FLIM format which provides quantitative readout of O(2) concentration within/near the cells, and live cell array devices for the monitoring of metabolic responses of individual suspension cells. Step by step procedures of typical experiments for each of these applications and troubleshooting guide are given.

Optical probes and techniques for O2 measurement in live cells and tissue

“…Phase fluorometry can also be affected by these factors, unless special signal processing algorithms are applied (see, e.g., [52]). Time-resolved fluorometry (TR-F) in the microsecond time domain allows better signal-to-noise ratio and more reliable and accurate O 2 quantification via measurement of probe emission decay [3, 81]. In a simplified format called rapid lifetime determination (RLD) [80, 82], emission intensity signals ( F 1 , F 2 ) are collected at two different delay times ( t 1 , t 2 ) after the excitation pulse, from which lifetime is calculated as:…”

“…With a proper calibration (e.g. measuring probe signal at several known pO 2 levels), fluorescence intensity images can be converted into [O 2 ] maps [81]. However, intensity calibrations are rather unstable due to significant probe photobleaching and signal drift under illumination (which should be minimised by all means), and affected by sample distortion (manipulation with cells or effector addition).…”

“…On a microscope with wide-field illumination, 2-D O 2 imaging can be realised using pulsed excitation with a suitable LED or laser delivering trains of ns-μs pulses at kHz frequency, and gated CCD camera operating in the microsecond time range [81]. Following each excitation pulse and a time delay (variable), emitted photons are collected by the camera over the measurement window time and integrated over a number of pulses to generate an intensity frame.…”

Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

“…(1) Various fluorescence and phosphorescence based O 2 -sensitive probes, including the complex platinum(II)-porphyrin dyes are widely used for imaging molecular oxygen in living organisms [17-19]. These probes exhibit a selective and reversible response to O 2 within the full physiological oxygen range (0 to 250 μM) combined with optimal photophysical properties (for example, high fluorescence brightness).…”

“…Among them, platinum(II)-porphyrin dyes are widely used for analyzing hypoxia-induced responses of mammalian cells [17-19]. Alternatively, the green fluorescent protein (GFP) and its variants can be applied as genetically encoded intracellular probes that are specifically expressed and can be selectively targeted within defined cells and tissues.…”