Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons
Michela Relucenti,
Giuseppe Familiari,
Orlando Donfrancesco,
Maurizio Taurino,
Xiaobo Li,
Rui Chen,
Marco Artini,
Rosanna Papa,
Laura Selan
Affiliations
Michela Relucenti
Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Via Alfonso Borelli 50, 00161 Rome, Italy
Giuseppe Familiari
Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Via Alfonso Borelli 50, 00161 Rome, Italy
Orlando Donfrancesco
Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Via Alfonso Borelli 50, 00161 Rome, Italy
Maurizio Taurino
Department of Clinical and Molecular Medicine, Unit of Vascular Surgery, Sant’Andrea Hospital, Sapienza University of Rome, Via di Grottarossa 1039, 00189 Rome, Italy
Xiaobo Li
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210096, China
Rui Chen
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210096, China
Marco Artini
Department of Public Health and Infectious Diseases, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
Rosanna Papa
Department of Public Health and Infectious Diseases, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
Laura Selan
Department of Public Health and Infectious Diseases, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.
