Bourges, A. C. | Search bioRxiv

bioRxiv2025

A non-toxic, user-friendly buffer that enhances green fluorophore performance in conventional and super-resolution imaging

Fluorophore brightness and photostability continue to be limiting factors in fluorescence microscopy. Several buffers that enhance these properties have been identified to date, though they do not work for all dyes, may require harmful components, or have limited storage lives. We have identified a simple, in-expensive, and non-toxic buffer that strongly enhances the brightness and resistance to photodestruction of green emissing synthetic fluorophores, using potassium iodide as a main photostabilizing additive and MgCl2 as enhancer for binding kinetics in DNA-PAINT super resolution imaging. We show that this AN-ice buffer can deliver an up to 30% improvement in fluorescence brightness and 5x more detected events in DNA-PAINT imaging. Furthermore, the same effect can be used to strongly increase the photobleaching resistance of all the green dyes tested for conventional imaging.

biochemistry↗

bioRxiv2019

Quantitative high-resolution imaging of live microbial cells at high hydrostatic pressure

The majority of the Earths microbial biomass exists in the Deep Biosphere, in the deep ocean and within the Earths crust. While 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 pressure on the Earths surface. The mechanisms of pressure adaptation are also important to 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 2-photon scanning microscope system allowing for the first time, 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 (sN&B) and Raster scanning Imaging Correlation Spectroscopy (RICS)) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in E. coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. SIGNIFICANCEThe majority of the Earths microbial biomass exists in high-pressure environments where pressures can reach over 100 MPa. The molecular mechanisms that allow microbes to flourish under such extreme conditions remain to be discovered. The high pressure, high resolution imaging system presented here revealed pressure dependent changes in metabolism and protein interactions in live microbial cells, demonstrating great promise for understanding deep life.

microbiology↗