Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment

Physiological Gut Oxygenation Alters GLP‐1 Secretion from the Enteroendocrine Cell Line STC‐1

peer-reviewed1 Scope Enteroendocrine cell lines are routinely assayed in simple buffers at ≈20% oxygen to screen foods for bioactives that boost satiety hormone levels. However, in vivo, enteroendocrine cells are exposed to different phases of food digestion and function at low oxygen concentration, ranging from 7.5% in the stomach to 0.5% in the colon–rectal junction. 2 Methods and results The objective of this study is to investigate the effect of physiologically relevant O2 concentrations of the gut on the production and secretion of the satiety hormone, glucagon‐like peptide 1 (GLP‐1), from the murine enteroendocrine cell line, secretin tumor cell line (STC‐1), in response to dairy macronutrients as they transit the gut. GLP‐1 exocytosis from STC‐1 cells is influenced by both oxygen concentration and by individual macronutrients. At low oxygen, STC‐1 cell viability is significantly improved for all macronutrient stimulations and cyclic adenosine monophosphate levels are dampened. GLP‐1 secretion from STC‐1 cells is influenced by both the phase of yogurt digestion and corresponding O2 concentration. Atmospheric oxygen at 4.5% combined with upper gastric digesta, which simulates ileum conditions, yields the highest GLP‐1 response. 3 Conclusion This demonstrates the importance of considering physiological oxygen levels and food digestion along gastrointestinal tract for reliable in vitro analysis of gut hormone secretion

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

Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

Synopsis Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O-2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O-2. A decrease in iO(2) (intracellular 02) to 0-10 mu M, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1 alpha and HIF-2 alpha, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO(2) nor HIF-alpha stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-alpha stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments

Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential

Background: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. Methods: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. Results: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in ‘energised’ negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. Conclusions: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. General significance: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies

Investigation of polyviologens as oxygen indicators in food packaging

A triggered oxygen indicator, formulated from a combination of electrochrome, titanium dioxide and EDTA, was evaluated for use in modified atmosphere packaging. Methylene blue was not an ideal electrochrome due to its slow reduction to the leuco form and fast subsequent oxidation by oxygen present at low concentrations, >0.1%. Polyviologen electrochromes showed much faster reduction after exposure to UV light. Thionine and 2,2?-dicyano-1,1?-dimethylviologen dimesylate, which have more anodic reduction potentials compared to methylene blue, can be used to produce oxygen indicators with decreased sensitivity to oxygen. These indicators can be used to detect oxygen even when levels increase up to 4.0%

Modeling the dynamics of hypoxia inducible factor-1α (HIF-1α) within single cells and 3D cell culture systems

HIF (hypoxia inducible factor) is an oxygen-regulated transcription factor that mediates the intracellular response to hypoxia in human cells. There is increasing evidence that cell signaling pathways encode temporal information, and thus cell fate may be determined by the dynamics of protein levels. We have developed a mathematical model to describe the transient dynamics of the HIF-1α protein measured in single cells subjected to hypoxic shock. The essential characteristics of these data are modeled with a system of differential equations describing the feedback inhibition between HIF-1α and prolyl hydroxylases (PHD) oxygen sensors. Heterogeneity in the single-cell data is accounted through parameter variation in the model. We previously identified the PHD2 isoform as the main PHD sensor responsible for controlling the HIF-1α transient response, and make here testable predictions regarding HIF-1α dynamics subject to repetitive hypoxic pulses. The model is further developed to describe the dynamics of HIF-1α in cells cultured as 3D spheroids, with oxygen dynamics parameterized using experimental measurements of oxygen within spheroids. We show that the dynamics of HIF-1α and transcriptional targets of HIF-1α display a non-monotone response to the oxygen dynamics. Specifically we demonstrate that the dynamic transient behaviour of HIF-1α results in differential dynamics in transcriptional targets

A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses

Stem cells and the niche in which they reside feature a complex microenvironment with tightly regulated homeostasis, cell-cell interactions and dynamic regulation of metabolism. A significant number of organoid models has been described over the last decade, yet few methodologies can enable single cell level resolution analysis of the stem cell niche metabolic demands, in real-time and without perturbing integrity. Here, we studied the redox metabolism of Lgr5-GFP intestinal organoids by two emerging microscopy approaches based on luminescence lifetime measurement - fluorescence-based FLIM for NAD(P)H, and phosphorescence-based PLIM for real-time oxygenation. We found that exposure of stem (Lgr5-GFP) and differentiated (no GFP) cells to high and low glucose concentrations resulted in measurable shifts in oxygenation and redox status. NAD(P)H-FLIM and O-2-PLIM both indicated that at high 'basal' glucose conditions, Lgr5-GFP cells had lower activity of oxidative phosphorylation when compared with cells lacking Lgr5. However, when exposed to low (0.5 mM) glucose, stem cells utilized oxidative metabolism more dynamically than non-stem cells. The high heterogeneity of complex 3D architecture and energy production pathways of Lgr5-GFP organoids were also confirmed by the extracellular flux (XF) analysis. Our data reveals that combined analysis of NAD(P)H-FLIM and organoid oxygenation by PLIM represents promising approach for studying stem cell niche metabolism in a live readout