A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Abstract

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF.

Mucus from human bronchial epithelial cultures: rheology and adhesion across length scales

“…Then the HPF specimen carrier with frozen mucus was inserted into the preparation chamber (Quorum PP3000T). For insertion, specimen shuttle cryo stubs and HPF specimen carrier adapter were used following the protocol from Payre et al [37]. After a fast transfer under vacuum in the preparation chamber, the samples were fractured at −140°C, sublimed at −95°C during 30 min and then coated by platinum sputtering.…”

How an organogelator can gelate water: gelation transfer from oil to water induced by a nanoemulsion

“…For insertion, specimen cryo shuttles and stubs with the right HPF specimen carrier adapter were used in accordance with the description in the publication by Payre B. and coll. 56 After quick transfer under vacuum into the preparation chamber, the samples were fractured at À140 1C, sublimed at À95 1C for 30 min and then coated using platinum sputtering. Lastly, they were transferred to the cryo-SEM Quanta 250 FEG chamber and kept at À140 1C for observation with an FEI Quanta 250 FEG scanning electron microscope at an accelerating voltage of 5 kV.…”