The principle of gravity-inertial orientation

The important problem of gyrocompass without drift creation is considered in this article. The designing device allows to define the accurate information about mobile and stationary objects orientation in the case of long-term (months, years) absence of the determining the cardinal direction possibility. This article focuses on the idea of gyrocompass without drift creation, which works on the principle of gravity-inertial orientation at the theoretical level

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

Luminescence lifetime imaging of three-dimensional biological objects

ABSTRACT A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein–protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.</jats:p

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

Intermittent Development of Central Place Systems: The Dynamics of Unification and Breakup

Relevance. The development of central place theory has been hindered by its static nature, as it fails to capture transitions between equilibrium states in central place systems. This long-standing problem remains unsolved since the theory's inception 90 years ago. This article presents a solution by examining the cases of system unification and system breakup, where previously independent systems merge or split.Research objective: The study aims to identify the conditions under which central place systems resume continual development following revolutionary transformations in their structure.Data and methods: The research analyzes census data from India (1947-2011) and Yemen (1973-2004) using equations based on the axioms of central place theory. The study also considers isostatic equilibrium as the foundation of central place system structures.Results: The effect of intermittence on the steady evolution of a central place system diminishes rapidly after the unification of two independent systems. In contrast, the adaptation of elements from a previously unified system to new conditions, including reinstating the former hierarchy and spatial structure, takes significantly longer after a system breakup. The study introduces a novel perspective, highlighting that the unification of central place systems tends to lead to progress, whereas the breakup of a unified system results in degradation.Conclusions: The true benefit to a central place system lies not solely in achieving isostatic equilibrium but in maintaining a secure and optimal structure. While these concepts share similarities, they may appear more distinct when examining the central place system as a whole. Equilibrium represents an optimal state for individual hierarchy levels rather than the entire system

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

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