Imaging of oxygen and hypoxia in cell and tissue samples

Abstract

Molecular oxygen (O₂) is a key player in cell mitochondrial function, redox balance and oxidative stress, normal tissue function and many common disease states. Various chemical, physical and biological methods have been proposed for measurement, real-time monitoring and imaging of O₂ concentration, state of decreased O₂ (hypoxia) and related parameters in cells and tissue. Here, we review the established and emerging optical microscopy techniques allowing to visualize O₂ levels in cells and tissue samples, mostly under in vitro and ex vivo, but also under in vivo settings. Particular examples include fluorescent hypoxia stains, fluorescent protein reporter systems, phosphorescent probes and nanosensors of different types. These techniques allow high-resolution mapping of O₂ gradients in live or post-mortem tissue, in 2D or 3D, qualitatively or quantitatively. They enable control and monitoring of oxygenation conditions and their correlation with other biomarkers of cell and tissue function. Comparison of these techniques and corresponding imaging setups, their analytical capabilities and typical applications are given.

Keywords: FLIM; Fluorescence and phosphorescence-based probes; Fluorescence microscopy; Hypoxia; Live cell and tissue imaging; Oxygen microscopy; PLIM.

Fig. 1

Schemes and signal readouts in optical O₂ and hypoxia imaging (see also Table 1). a Redox-sensitive indicators (e.g., pimonidazole) coupled with specific antibody staining of fixed cells/tissue. b Redox-sensitive fluorescent hypoxia stains and fluorescent proteins expressed under HRE promoter. Cells and tissues can be imaged live and fluorescence of different regions of interest (ROI) compared to assess the degree of hypoxia. c Fluorescent proteins with maturation rate sensitive to O₂, e.g., UnaG-based [27]. Green fluorescent protein has O₂-independent maturation, while the red is not produced under hypoxia. Signal ratio can report on hypoxia. d Red-shifted O₂-dependent fluorescence of GFP induced by photoactivation [49]. The ratio between red and green fluorescence reports on hypoxia (reversible changes). e–g Quenched phosphorescence and delayed fluorescence-based O₂ probes. e O₂ probes reversibly change brightness (increases at hypoxia), can be used in semi-quantitative mode shown in b. f Ratiometric O₂ probes have two emission bands (e.g., blue and red [, –52]), which allow signal normalization and O₂ quantification. g One or two-photon PLIM allows accurate O₂ quantification, from measured emission decays and calculated phosphorescence lifetimes (O₂ sensitive). Calibration, frequently shown as Stern–Volmer plots, can be linear [53] or non-linear (fitted with the two-site model [54]). PLIM can visualize tissue O₂ concentration maps, subcellular gradients, and their dynamics