Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition

TY - JOUR

T1 - Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition

AU - Alhede, Morten

AU - Qvortrup, Klaus

AU - Liebrechts, Ramon

AU - Høiby, Niels

AU - Givskov, Michael

AU - Bjarnsholt, Thomas

N1 - © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

PY - 2012/7

Y1 - 2012/7

N2 - Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.

AB - Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.

U2 - 10.1111/j.1574-695x.2012.00956.x

DO - 10.1111/j.1574-695x.2012.00956.x

M3 - Journal article

C2 - 22429654

SN - 2049-632X

VL - 65

SP - 335

EP - 342

JO - Pathogens and Disease

JF - Pathogens and Disease

IS - 2

ER -