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The sample preparation for cryo-SEM: the real ultrastructure of microbial biofilm or just artifacts?
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SYSNO ASEP 0465339 Document Type C - Proceedings Paper (int. conf.) R&D Document Type Conference Paper Title The sample preparation for cryo-SEM: the real ultrastructure of microbial biofilm or just artifacts? Author(s) Hrubanová, Kamila (UPT-D) RID, SAI, ORCID
Skoupý, Radim (UPT-D) RID, ORCID, SAI
Nebesářová, Jana (BC-A) RID, ORCID
Růžička, F. (CZ)
Krzyžánek, Vladislav (UPT-D) RID, ORCID, SAINumber of authors 5 Source Title EMC2016. The 16th European Microscopy Congress. Proceedings. - Oxford : Wiley, 2016 - ISBN 9783527808465 Pages s. 203-204 Number of pages 2 s. Publication form Online - E Action EMC2016. European Microscopy Congress /16./ Event date 28.08.2016 - 02.09.2016 VEvent location Lyon Country FR - France Event type WRD Language eng - English Country GB - United Kingdom Keywords biofilm ; Cryo-SEM ; HPF Subject RIV JA - Electronics ; Optoelectronics, Electrical Engineering Subject RIV - cooperation Electronics ; Optoelectronics, Electrical Engineering R&D Projects GA14-20012S GA ČR - Czech Science Foundation (CSF) GA16-12477S GA ČR - Czech Science Foundation (CSF) Institutional support UPT-D - RVO:68081731 ; BC-A - RVO:60077344 DOI 10.1002/9783527808465.EMC2016.6907 Annotation 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. Workplace Institute of Scientific Instruments Contact Martina Šillerová, sillerova@ISIBrno.Cz, Tel.: 541 514 178 Year of Publishing 2017
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