Ruslan I. Dmitriev

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

In recent years, significant progress has been achieved in the sensing and imaging of molecular oxygen (O2) in biological samples containing live cells and tissue. We review recent developments in the measurement of O2 in such samples by optical means, particularly using the phosphorescence quenching technique. The main types of soluble O2 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 O2 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 O2 consumption rate, sample oxygenation, sensing of intracellular O2, metabolic assessment of cells, and O2 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.

Assessment of Cellular Oxygen Gradients with a Panel of Phosphorescent Oxygen-Sensitive Probes

The supply of oxygen (O(2)) to respiring tissue, cells, and mitochondria regulates metabolism, gene expression, and cell fate. Depending on the cell type and mitochondrial function, O(2) gradients between extra- and intracellular compartments may vary and play important physiological roles such as the regulation of activity of prolyl hydroxylases and adaptive responses to hypoxia. Here we present a new methodology for the analysis of localized O(2) gradients in cultures of adherent cells, using three phosphorescent Pt-porphyrin based probes with different localization. One new O(2) probe targeted to the cell membrane was developed and used together with existing MitoXpress and Nano2 probes to monitor mean pericellular (PC), extracellular (EC), and intracellular (IC) O(2) concentrations, respectively. Mouse fibroblasts and neuronal PC12 cells cultured in standard microplates were stained with probes and measured on a commercial time-resolved fluorescence reader in phosphorescence lifetime mode. Respiring cells exposed to various levels of atmospheric O(2) showed differences in oxygenation of their IC, PC, and EC compartments. Experiments with different cell numbers and modulation of respiration activity demonstrated that these gradients are dynamic and regulated by the O(2) diffusion and consumption rate. The new method facilitates the assessment of such gradients.

Imaging of neurosphere oxygenation with phosphorescent probes

Multicellular spheroids are useful models of mammalian tissue for studies of cell proliferation, differentiation, replacement therapies and drug action. Having a size of 100-500 μm they mimic in vivo micro-environment and characteristic gradients of O2, pH and nutrients. We describe the use of cell-penetrating O2 probes based on phosphorescent Pt-porphyrins to perform high-resolution 2D and 3D mapping of O2 in spheroid structures by live cell fluorescence imaging technique. Optimised procedures for preparation of neurospheres from cortical neural cells isolated from embryonic rat brain, their staining with the phosphorescent O2 probes NanO2 and MM2 and subsequent analysis of oxygenation on different live cell imaging platforms, including widefield and confocal phosphorescence lifetime imaging microscopy (PLIM), conventional confocal and two-photon ratiometric intensity based O2 detection are presented. This is followed by a series of physiological experiments in which oxygenation patterns of the neurospheres are correlated with culturing conditions (atmospheric hypoxia and hyperoxia, size, growth factors), distribution of stem cells, mature neurons and astrocytes, HIF-2α stabilisation and responses to metabolic stimulation. The O2 imaging method allows multiplexing with many conventional fluorescent probes to perform multi-parametric imaging analysis of cells in 3D microenvironment. It can be applied to other types of spheroids and 3D tissue models.

Small molecule phosphorescent probes for O2 imaging in 3D tissue models

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 μM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 μs in air-saturated to 57 μs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.

Intracellular oxygen-sensitive phosphorescent probes based on cell-penetrating peptides

Probing of molecular oxygen in mammalian cells is important for the analysis of mitochondrial function, metabolic responses, and energetic status of the cells. We describe a new panel of intracellular O(2)-sensitive probes based on phosphorescent porphyrin dyes conjugated to cell-penetrating peptides. The probes comprising the uncharged derivatives of Pt(II)-coproporphyrin I covalently linked to positively charged TAT-derived peptides are shown to effectively load live mammalian cells without any transfection reagents. The probes work well with all cell types tested, show similar subcellular localization, and produce characteristic responses to cell stimulation with mitochondrial uncouplers and inhibitors. They provide a simple and versatile tool for O(2) monitoring in live cells and in tissue, and an alternative to the existing O(2) probes which require facilitated transport into the cell.

Complexes of IrIII-Octaethylporphyrin with Peptides as Probes for Sensing Cellular O2

IrIII–porphyrins are a relatively new group of phosphorescent dyes that have potential for oxygen sensing and labeling of biomolecules. The requirement of two axial ligands for the IrIII ion permits simple linkage of biomolecules by a one‐step ligand‐exchange reaction, for example, using precursor carbonyl chloride complexes and peptides containing histidine residue(s). Using this approach, we produced three complexes of IrIII–octaethylporphyrin with cell‐penetrating (Ir1 and Ir2) and tumor‐targeting (Ir3) peptides and studied their photophysical properties. All of the complexes were stable and possessed bright, long‐decay (unquenched lifetimes exceeding 45 μs) phosphorescence at around 650 nm, with moderate sensitivity to oxygen. The Ir1 and Ir2 complexes showed positive staining of a number of mammalian cell types, thus demonstrating localization similar to endoplasmic reticulum and ATP‐ and temperature‐independent intracellular accumulation (direct translocation mechanism). Their low photo‐ and cytotoxicity allows intracellular oxygen to be probed.

Cell-Penetrating Conjugates of Coproporphyrins with Oligoarginine Peptides: Rational Design and Application for Sensing Intracellular O2

A panel of phosphorescent oligoarginine conjugates of tetracarboxylic Pt(II)-coproporphyrin I dye (PtCP), monosubstituted with long peptides or tetra-substituted with short peptides and having different linkers and peripheral groups, is described. Their photophysical properties, cell loading efficiency, and mechanisms of transport into the cell were investigated and compared. The conjugates were seen to rely on endocytotic mechanisms of cell entry, which are different from that of the unconjugated oligoarginine peptide, and show diverse patterns of intracellular distribution. On the basis of this study, the tetra-substituted PtCP conjugate displaying whole cell distribution was selected for the sensing of intracellular O(2). This probe has been tested in biological experiments on a fluorescence plate reader, including the monitoring of in situ oxygenation of respiring cells and their responses to metabolic stimulation. Similar conjugates of the phosphorescent Pd(II)-coprorphyrin and fluorescent coproporphyrin-ketone were also synthesized and assessed for the sensing of low levels intracellular O(2) and ratiometric pH-sensing, respectively. The results produced and the structure-activity relationships determined can facilitate the rational design of new bioconjugates of porphyrin dyes tailored to specific applications.

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O2 levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O2 imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O2 in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O2 levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O2 in 3D tissue models.

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor.

Research on cell‐penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans‐activator of transcription (TAT)‐derived peptides and, to a lesser extent, on proline‐rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen‐sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15–24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH‐SY5Y and HeLa, via cell‐type‐dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time‐resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen‐sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O2 levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.

Intracellular probes for imaging oxygen concentration: how good are they?

In the last decade a number of cell-permeable phosphorescence based probes for imaging of (intra)cellular oxygen (icO2) have been described. These small molecule, supramolecular and nanoparticle structures, although allowing analysis of hypoxia, local gradients and fluctuations in O2, responses to stimulation and drug treatment at sub-cellular level with high spatial and temporal resolution, differ significantly in their operational performance and applicability to different cell and tissue models. Here we discuss and compare these probes with respect to their staining efficiency, brightness, photostability, toxicity, cell specificity, compatibility with different cell and tissue models, and analytical performance. Merits and limitations of particular probes are highlighted and strategies for development of new high-performance O2 imaging probes defined. Key application areas in hypoxia research, stem cells, cancer biology and tissue physiology are also discussed.