A modified ‘NanoSuit®’ preserves wet samples in high vacuum: direct observations on cells and tissues in field-emission scanning electron microscopy

Abstract

Although field-emission scanning electron microscopy (FE-SEM) has proven very useful in biomedical research, the high vacuum required (10−3 to 10−7 Pa) precludes direct observations of living cells and tissues at high resolution and often produces unwanted structural changes. We have previously described a method that allows the investigator to keep a variety of insect larvae alive in the high vacuum environment of the electron microscope by encasing the organisms in a thin, vacuum-proof suit, the ‘NanoSuit®'. However, it was impossible to protect wet tissues freshly excised from intact organisms or cultured cells. Here we describe an improved ‘NanoSuit' technique to overcome this limitation. We protected the specimens with a surface shield enhancer (SSE) solution that consists of glycerine and electrolytes and found that the fine structure of the SSE-treated specimens is superior to that of conventionally prepared specimens. The SSE-based NanoSuit affords a much stronger barrier to gas and/or liquid loss than the previous NanoSuit did and, since it allows more detailed images, it could significantly help to elucidate the ‘real' organization of cells and their functions.

Utility of Scanning Electron Microscopy Elemental Analysis Using the ‘NanoSuit’ Correlative Light and Electron Microscopy Method in the Diagnosis of Lanthanum Phosphate Deposition in the Esophagogastroduodenal Mucosa

“…(4) Imaging occurs in a wet state approximating natural conditions. Less structural damage has been proven using the NanoSuit-CLEM method compared to the conventional method for SEM observation [18,19].…”

“…First, the cover glass was removed with xylene and the section on the slide was re-hydrated using a drop of surface shield enhancer (SSE) solution. The stock solution of SSE consisted of sucrose, fructose, and sodium chloride dissolved in distilled water, to which citric acid and sodium glutamate (pH 7.4) were then added [18]. The resulting aqueous solution was mixed with glycerine at a ratio of 1:2.…”

“…SSE was diluted 20-fold in ethanol. Sections were subsequently spin-coated by centrifugation at 2000 rpm for 15 sec to remove excess of the SSE solution [18]. The specimens were then directly introduced into the SEM to form the NanoSuit following irradiation of the electron beam.…”

“…We previously reported that polymerization of a natural extracellular substance on the outer surface of animals by electron beam or plasma irradiation resulted in the formation of a nano-scale layer, called the 'NanoSuit', which enables small animals to stay alive under SEM observation [17]. We have recently developed the new correlative light and electron microscopy (CLEM) method using NanoSuit (NanoSuit-CLEM method), which enables not only CLEM but also EDS analysis [18,19]. The sections of formalin-fixed paraffin-embedded (FFPE) tissue on glass slides are easily observed both by light and electron microscope showing less structural damage and weight loss than in the case of conventional methods for SEM observation [18,19].…”

“…We have recently developed the new correlative light and electron microscopy (CLEM) method using NanoSuit (NanoSuit-CLEM method), which enables not only CLEM but also EDS analysis [18,19]. The sections of formalin-fixed paraffin-embedded (FFPE) tissue on glass slides are easily observed both by light and electron microscope showing less structural damage and weight loss than in the case of conventional methods for SEM observation [18,19]. This method requires only several minutes to observe the pathological slides after the cover slips are removed, and does not require additional treatment, such as osmium conductive metal coating.…”