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

Abstract

In recent years, significant progress has been achieved in the sensing and imaging of molecular oxygen (O(2)) in biological samples containing live cells and tissue. We review recent developments in the measurement of O(2) in such samples by optical means, particularly using the phosphorescence quenching technique. The main types of soluble O(2) sensors are assessed, including small molecule, supramolecular and particle-based structures used as extracellular or intracellular probes in conjunction with different detection modalities and measurement formats. For the different O(2) sensing systems, particular attention is paid to their merits and limitations, analytical performance, general convenience and applicability in specific biological applications. The latter include measurement of O(2) consumption rate, sample oxygenation, sensing of intracellular O(2), metabolic assessment of cells, and O(2) imaging of tissue, vasculature and individual cells. Altogether, this gives the potential user a comprehensive guide for the proper selection of the appropriate optical probe(s) and detection platform to suit their particular biological applications and measurement requirements.

Fig. 1

Different signal profiles revealed with the O₂ sensing probes. a Microbial respiration/growth of E. coli measured in phosphorescence intensity mode. The number of cells in the original sample (cfu/g) is determined from the time required to reach signal threshold [121]. b Respiration profiles of eukaryotic cells measured in RLD mode: wild-type (WT) mouse embryonic fibroblasts, knock-out (KO) cells deficient with Krebs cycle enzyme and blank control. OCRs are calculated from the slope of probe signal [57]. c Profiles of oxygenation for the respiring and non-respiring PC12 cells under mild atmospheric hypoxia (8% pO₂). d Changes in icO₂ in respiring PC12 cells upon the addition of uncoupler (FCCP), inhibitor (AntA) and mock control (DMSO), at 20.9% pO₂. Arrow indicates the time of effector addition. e Oxygenation of MEF cells grown at different densities in a microfluidic chip Ibidi^®, measured under static conditions. Flushing the chamber with fresh medium (at the start and at arrow) causes reoxygenation and subsequent deoxygenation of the cells. a, b were generated with ecO₂ probe MitoXpress^™ [80]; c–e with pre-calibrated icO₂ probes [61, 71]

Fig. 2

Hypoxia workstation with optical O₂ probes and sensors. a Glove-box with controlled atmosphere (adjustable O₂), temperature and humidity, in which cultured cells, accessories and measurement equipment are placed. b Solid-state O₂ sensors placed inside the glove box are read with a hand-held scanner from the outside. c Tissue culture flask with built-in O₂ sensor dot that can also be measured with the scanner. d Microplate with cultured cells to which an O₂-sensing probe is added. e Live cell O₂-imaging system—fluorescent microscope with FLIM capabilities. f TR-F microplate reader which can read the IC and EC probes and measure OCR and icO₂ in a microplate with respiring samples; this instrument is also equipped with O₂/CO₂ control of microplate compartment