Application of Environmental Scanning Electron Microscope-Nanomanipulation System on Spheroplast Yeast Cells Surface Observation

Abstract

The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD800 absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl2 (CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD800 absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.

Informative three-dimensional survey of cell/tissue architectures in thick paraffin sections by simple low-vacuum scanning electron microscopy

“…Scanning electron microscopy (SEM) provides three-dimensional information of specimen surfaces by collecting electrons reflected from the surface (backscattered electrons, BSE) and electrons forced out of the surface (secondary electrons, SE). Low-vacuum SEM allows for the BSE and/or SE imaging of non-conductive biological samples 4 – 7 because the negative charge accumulations on the non-conductive materials can be eliminated with the positive ions in residual gas molecules 8 , 9 .…”

Quasi‐Newtonian Environmental Scanning Electron Microscopy (QN‐ESEM) for Monitoring Material Dynamics in High‐Pressure Gaseous Environments

“…Environmental scanning electron microscopy (ESEM) has emerged as a powerful tool to characterize a diversity of samples including both wet and insulating materials in a straightforward manner, that is, no significant specimen preparation or metallic layer coating is required. [1][2][3] Such an extraordinary feature has boosted the applications of ESEM in many fields such as colloidal dispersions, [4][5][6][7] chemical reactions, [8][9][10] biological specimen, [11][12][13] phase changes, [14][15][16] micromechanics, [17] and complex fluids. [18] Generally, the success of ESEM relies heavily on the participation of gas molecules in the imaging process: electrons that escape from non-conducting sample surfaces travel through the gas and produce a cascaded amplification of electrons by ionizing the gas molecules, resulting in positive ions that can drift down to the sample and thus compensate the accumulated surface charges.…”

Three-dimensional fine structures in deep fascia revealed by combined use of cryo-fixed histochemistry and low-vacuum scanning microscopy

“…This allows for a simple three-dimensional (3D) cell/tissue architecture survey method where the sample is embedded in the same paraffin sections as those used for histological analysis, such as Hematoxylin–Eosin (HE) staining. In general, SEM provides 3D information about specimen surfaces by collecting backscattered electrons (BSE) reflected from the surface, as well as secondary electrons (SE) which are forced out of the surface 25 – 28 . While conventional SEM is capable of imaging the 3D microstructure of samples with high resolution, it is unsuitable for non-conductive paraffin sections because of the negative charge accumulation.…”