Correlative Light-Environmental Scanning Electron Microscopy of Plasma Membrane Efflux Carriers of Plant Hormone Auxin

Abstract

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

Decellularized Pancreatic Tail as Matrix for Pancreatic Islet Transplantation into the Greater Omentum in Rats

“…A morphological study of the ECM skeletons was realized with an advanced environmental scanning electron microscope (A-ESEM) [ 27 ], in combination with the low temperature method (LTM) [ 28 , 29 ]. Briefly, tissue samples were fixed in buffered 4% formaldehyde overnight.…”

Slip Flow Analysis in an Experimental Chamber Simulating Differential Pumping in an Environmental Scanning Electron Microscope

“…The environmental scanning electron microscope (ESEM) allows, due to the presence of gas pressures in the order of units of thousands of Pa in the specimen chamber, the observation of electrically non-conductive or semiconductive samples without charge artifacts [ 1 ], the observation of sensitive biological [ 2 ] and polymeric [ 3 ] samples without damage in their native state by special methods [ 1 ], or the study of these samples in dynamic in situ experiments [ 3 ]. Signal electrons are detected in ESEMs using special ionization [ 4 , 5 ] or scintillation detectors [ 6 , 7 , 8 ], whose images can be correlated with light microscope images [ 9 ].…”

Analysis of gas thermal conductivity at low pressures using a mathematical-physical model

“…Due to the presence of a gas pressure in the order of units to thousands of Pa in the sample chamber, the environmental scanning electron microscope (ESEM) allows to observe electrically non-conductive or semiconductive samples without charge artifacts (Danilatos), or to observe sensitive biological [4] and polymeric [5] samples in their native state without any damage with special methods [6], or to study these samples in dynamic in-situ experiments [5]. Signal electrons are detected in the ESEM using special ionization [7,8] or scintillation detectors [9,10,11], whose images can be correlated with light microscope images [12].…”