Table 1.

Main types of probes used in fluorescence-based imaging of cell and tissue O₂ and hypoxia

No.	Probe type	Operation principle	Typical procedure	Advantages/limitations	References
Indirect probes and techniques for visualization of hypoxia
P1	Redox-sensitive nitroimidazole derivatives	Penetrates cells and tissue and is converted at low O2 by nitroreductase enzyme. Product is detected by antibody staining and fluorescence microscopy	Systemic administration (in blood or medium), accumulation, conversion in cells, tissue sectioning, fixation, immunofluorescence, microscopy imaging	Small molecule probes, work in cells and animals, use basic fluorescence microscope Endpoint, qualitative (threshold type), dead tissue	[23–25]
P2	Redox dye-based fluorescent hypoxia-sensitive chemical probes	Cell-permeable precursor dyes, which generate fluorescence upon enzymatic conversion at low O2. Work similar to P1, but do not require fixation and antibody staining	Administration, incubation, (live) fluorescence imaging	Live imaging of whole animals Indirect, repetitive Complex signal dynamics, qualitative (threshold type), not always reversible	[20, 26]
P3	Fluorescent protein reporter-based systems	Expression of an endogenous fluorescent protein controlled by a hypoxia-responsive promoter (e.g., HIF). Fluorescence is generated in hypoxic zones	DNA transfection (transient or with generation of stable transgenic cell lines or animals), incubation, fluorescence imaging	Endogenous probe, live, high-resolution imaging, 2D and 3D. Broadly compatible Qualitative, threshold type. Complex signal dynamics. Not always reversible. Maturation of fluorescent protein can be affected by O2. Complex assay (UnaG-based system) [27]	[27–31]
P4	Prolyl hydroxylase activity-dependent biosensor constructs	Endogenously expressed FRET biosensor (ProCY) responsive to prolyl hydroxylase activity	DNA transfection (transient or with generation of stable transgenic cells lines or animals), incubation, fluorescence imaging	Endogenous ratiometric probe, live high-resolution imaging, 3D capabilities Semi-quantitative. Complex signal dynamics	[32]
P5	O2-dependent fluorescent proteins	Endogenously expressed fluorescent protein (or FRET-based multi-protein biosensor), based on GFP, YFP or DsRed with O2-dependent maturation/folding	DNA transfection (transient or with generation of stable transgenic cells lines or animals), incubation, fluorescence imaging	Endogenous probe, live and post-mortem imaging, 2D, 3D and whole animals. ‘Memorize’ cellular O2 levels [33] Complex signal dynamics, The effects of protein overproduction on cell function are poorly studied	[33–35]
Direct and reversible optical sensing of O2 concentration/partial pressure
P6	Delayed fluorescence of endogenously overproduced protoporphyrin IX (PPIX)	Dynamic quenching of endogenous PPIX-delayed fluorescence, which is overproduced in mitochondria after 5-aminolevulinic acid (5-ALA) administration	Administration of 5-ALA to cells or tissue, incubation, live phosphorescence lifetime imaging microscopy (PLIM)	Natural fluorophore with mitochondrial localization, quantitative, real-time, so far used for point measurements Weak signals, long emission lifetimes. 5-ALA effects on cell metabolism are unstudied	[36–38]
P7	Intravascular (cell-impermeable) phosphorescent probes	Dynamic quenching of an exogenous cell-impermeable phosphor with complex supramolecular (dendrimer) structure and strong two-photon excitability	Administration in blood stream, imaging of vascular and interstitial O2	Live, quantitative, real-time two-photon excited in vivo imaging. Stable O2 calibration in serum and some other biological fluids. 3D and combination with other blood flow and metabolism is also possible High probe doses upon systemic administration, short time window. Mostly applicable for imaging blood vessels and microcapillaries. Complex procedure and two-photon PLIM. Limited use with in vitro models	Reviewed in [12, 39]
P8	Cell-penetrating small molecule phosphors and conjugates	Dynamic quenching of an exogenous phosphor, which has intrinsic ability to efficiently stain cells or 3D tissue models. Various chemical groups such as carbohydrates, mitochondrial targeting groups or cell-penetrating peptides are used as delivery vectors	Probe addition to the growth medium or local administration in animal, incubation (1–16 h), imaging on fluorescence or PLIM microscopes	Live, quantitative, real-time imaging of intracellular O2 in 2D/3D models. Deep tissue staining, long retention times (days), local administration, different chemistries and spectra Moderate intensity signals. Interaction with biomolecules can alter O2 calibration (cell-specific calibration). Usually require PLIM mode and one-photon excitation. Some probes show toxicity	Reviewed in [12, 16–18]
P9	Phosphorescent nanosensors	Dynamic quenching of an exogenous phosphor embedded in cell-penetrating polymeric nanoparticles	Probe addition to the growth medium or local administration in animal, incubation (1–16 h), imaging on fluorescence or PLIM microscopes	Live, real-time, quantitative (ratiometric, PLIM) imaging of intracellular O2 in 2D/3D models. High brightness, also under two-photon excitation, low doses and impact on cell function Cell specificity, slow uptake (12–16 h), less efficient than P8 in 3D tissue staining. Can be toxic/invasive (> 100 nm size of nanosensors)	Reviewed in [12, 16–18, 40]
P10	Solid-state phosphorescent sensors: coated foils, microparticles and porous scaffolds	Dynamic quenching of the phosphorescent dye molecules embedded in a film coating, microparticles, microporous scaffold or related materials. Measures only peri- and extracellular O2	Sensor film or suspension of microparticles is applied or sprayed to cells or tissue surface and imaged In case of hybrid porous O2-sensing polymer scaffolds cells are grown within scaffold and then imaged using fluorescence of PLIM microscopes	Live, real-time, quantitative 2D or 3D imaging, no staining of the sample. Stable calibration, high-optical signals (intensity, ratiometric or PLIM readouts) Non-invasive, but can only image areas of contact or extracellular O2 (scaffolds)	[41, 42–44, 10, 19, 45, 46, 47, 48]