Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

Abstract

Papkovsky DB. Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging. Am J Physiol Regul Integr Comp Physiol 292: R1613-R1620, 2007. First published December 14, 2006; doi:10.1152/ajpregu.00707.2006.-The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptormediated Ca 2ϩ influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer. metalloporphyrin; mitochondrial function; uncoupling MOLECULAR OXYGEN IS A KEY substrate in aerobic biological systems, providing the terminal electron acceptor of the electron transport chain (7). The rate of oxygen consumption is closely regulated by the ATP/ADP ratio (24), while also being highly dependent on the concentration of available oxygen (31) and the action of signaling molecules such as NO (9, 37) and Ca 2ϩ (10, 23), which, in turn, are related to pathways of cell survival and death (22). Furthermore, the induction of cellular hypoxia results in the activation of a specific adaptive transcriptional response governed by the hypoxia inducible transcription factors (9). Oxygen consumption is, therefore, an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. It may be used to elucidate biochemical pathways of the cell and alterations in metabolism caused by various stimuli or disease states. Accordingly, there is a requirement for methods that can effectively monitor cellular oxygen levels and oxygen consumption rates.Optical schemes, based on the quenching by molecular oxygen of long-decay fluorescent and phosphorescent dyes, such as metalloporphyrins and ruthenium(II) complexes, have been under active development in recent years (32). The group of Wilson and coworkers (8, 47) ha...

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…On the other hand, it is clear that respiratory responses of individual cells can be more diverse and complex than bulk responses of cell populations. These aspects are outside the scope of this study and are the subject of a separate investigation using an i O 2 imaging method (29).…”

“…Thus far these parameters were not amenable to routine analysis. The well established O 2 respirometry technique (28) has limited applicability to suspension cell lines, low resolution power, and information content (end-point measurement), whereas the i O 2 imaging technique (29), although allowing detailed singlecell analyses, has a high level of complexity and low sample throughput. The new fluorescence-based methodology of sensing intracellular O 2 (30) addresses these limitations and provides simple, high throughput analysis of i O 2 gradients in cell populations and effects of various metabolic effectors and stimuli.…”

Molecular Imaging of Hypoxia

“…The average difference between melanoma and normal tissue was 10 mm Hg, and the lowest average tumor PO 2 was 8 mm Hg, higher than in many solid tumors. It is not likely that this approach will have widespread clinical value, but it should find a role in smallanimal studies and is finding an important role in cell biology (204) as new and more photostable chromophores are developed. The oxygen-quenching effect is rapidly reversible so that this assay is useful for observing rapid changes in oxygenation status, including reoxygenation.…”

Water-soluble cyclometalated platinum(ii) and iridium(iii) complexes: synthesis, tuning of the photophysical properties, and in vitro and in vivo phosphorescence lifetime imaging

“…1,4,11,12 As a result, advanced sensing platforms were developed to recognize various biologically relevant analytes, including (but not limited to) detection of cyanide ions, 13 biothiols 14 (including thiourea 15 ), b-galactosidase, 16 dopamine receptor, 17 or inhibitor of tumor necrosis factor-a. 18 Moreover, the sensitivity of emission parameters (intensity and lifetime) to oxygen concentration makes possible mapping of oxygen concentration in in vitro and in vivo experiments, 19,20 including quantitative measurements with the use not only emission intensity but also phosphorescence lifetime imaging (PLIM) techniques, [21][22][23][24][25] which provides considerably higher precision in oxygen biodistribution mapping. This turns traditional luminescence imaging into the instrument, which allows for functional analysis of physiological status of the targeted objects.…”