No abstract

The well-known saying of “Seeing is believing” became even more apt in biology when stimulated emission depletion (STED) nanoscopy was introduced in 1994 by the Nobel laureate S. Hell and coworkers. We presently stand at a juncture. Nanoscopy represented a revolution in fluorescence microscopy but now is a mature technique applied to many branches of biology, physics, chemistry, and materials science. We are currently looking ahead to the next generation of optical nanoscopes, to the new key player that will arise in the forthcoming years. This article gives an overview of the various cutting-edge implementations of STED nanoscopy and tries to shine a light into the future: imaging everything faster with unprecedented sensitivity and label-free.

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O₂. The ability of OS to manipulate O₂ may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.

BACKGROUND AND PURPOSEThe rod outer segments (OS) of the retina are specialized organelles where phototransduction takes place. The mitochondrial electron transport complexes I-IV, cytochrome c and FoF1-ATP synthase are functionally expressed in the OS disks. Here, we have studied the effect of some polyphenolic compounds acting as inhibitors of mitochondrial ATPase/synthase activity on the OS ectopic FoF1-ATP synthase. The mechanism of apoptosis in the OS was also investigated studying the expression of cytochrome c, caspase 9 and 3 and Apaf-1. EXPERIMENTAL APPROACHWe prepared OS from fresh bovine retinae. Semi-quantitative Western blotting, confocal and electron microscopy, and cytofluorimetry were used along with biochemical analyses such as oximetry, ATP synthesis and hydrolysis. KEY RESULTSResveratrol and curcumin plus piperine inhibited ATP synthesis and oxygen consumption in the OS. Epigallocatechin gallate and quercetin inhibited ATP hydrolysis and oxygen consumption in the OS. Malondialdehyde and hydrogen peroxide were produced in respiring OS in the presence of substrates. Cytochrome c was located inside the disk membranes. Procaspase 9 and 3, as well as Apaf-1 were expressed in the OS. CONCLUSIONS AND IMPLICATIONSThese polyphenolic phytochemicals modulated the FoF1-ATP synthase activity of the the OS reducing production of reactive oxygen intermediates by the OS ectopic electron transport chain. Polyphenols decrease membrane peroxidation and cytochrome c release from disks, preventing the induction of caspase-dependent apoptosis in the OS Such effects are relevant in the design of protection against functional impairment of the OS following oxidative stress from exposure to intense illumination.