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Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca 2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca 2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems., (© 2022. The Author(s).)

The majority of the Earth's microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth's crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth's surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)