The sample preparation for cryo-SEM: the real ultrastructure of microbial biofilm or just artifacts?

0465339 - ÚPT 2017 RIV GB eng C - Conference Paper (international conference)
Hrubanová, Kamila - Skoupý, Radim - Nebesářová, Jana - Růžička, F. - Krzyžánek, Vladislav
The sample preparation for cryo-SEM: the real ultrastructure of microbial biofilm or just artifacts?
EMC2016. The 16th European Microscopy Congress. Proceedings. Oxford: Wiley, 2016, s. 203-204. ISBN 9783527808465.
[EMC2016. European Microscopy Congress /16./. Lyon (FR), 28.08.2016-02.09.2016]
R&D Projects: GA ČR(CZ) GA14-20012S; GA ČR(CZ) GA16-12477S
Institutional support: RVO:68081731 ; RVO:60077344
Keywords : biofilm * Cryo-SEM * HPF
Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering
http://onlinelibrary.wiley.com/doi/10.1002/9783527808465.EMC2016.6907/pdf

The cryo-scanning electron microscopy (cryo-SEM) belongs to reputable techniques in electron microscopy of hydrated samples such as biofilms. The crucial steps of the cryo-preparation techniques are primarily the cryo-fixation and partial sublimation of ice contamination caused during the transfer of the sample to the cryo-high-vacuum preparation chamber where the sublimation process is performed; optionally the freeze-fracturing or coating by metal sputtering or carbon evaporation can be applied. In the case of cryo-fixation, an effort is to keep the frozen biofilm in the form nearby its native state. One of the simplest cryo-fixation techniques is a plunging of the biofilm on a substrate into a liquid cryogen. However, the plunging into a liquid nitrogen or even liquid ethane/propane is sufficient for fixation of very thin layers of biofilm (no more than a few micrometers in thickness) because it is very difficult to achieve enough cooling rates to produce amorphous ice in the sample due to the Leidenfrost effect. Moreover, we show that the cryo-fixation into liquid nitrogen can lead to significant lateral macro-segregation of both bacteria and extracellular polymeric substances (EPS), where plunging into liquid ethane leads to micro-segregation of EPS and macro-segregation of bacteria. Substantially more effective cooling can be achieved by increasing the pressure during exposure to the liquid cryogen. This can be performed for example by the high-pressure freezing (HPF) technique. It was proved that cryo-fixed biofilms by HPF show significantly improved preservation of bacterial ultrastructure and biofilm organization.
Permanent Link: http://hdl.handle.net/11104/0263955