Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

Abstract

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.

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“…Also, the biofilm may be discontinuous and may not be detected on the collected fragments. The cryofixation SEM variant provides better preservation of extracellular polymeric substances [ 56 ].…”

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“…All samples were dried in a critical point desiccator (Emitech K850, Kent, UK). Then, about 15–20 nm gold spray coating was performed with the Balzers SCD 030 (New York, USA) and the images were achieved using the Supra 40 ZEISS (EHT = 5.00 kV, WD = 22 mm, detector in the lens) (Berlin, Germany) [ 28 ].…”

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“…After the growth, SEM stubs were washed in 0.1 M phosphate buffer pH 7.4 (PB) and fixed in 2.5% glutaraldehyde in 0.1 M PB buffer. Samples were washed overnight in PB and postfixed with a mixture of 2% OsO 4 and 0.2% Ruthenium Red, for 1 h at room temperature [ 44 , 45 , 46 ]. Samples were then washed for 30 min with H 2 O, the excess of water was dried carefully with filter paper and then the samples were mounted onto the specimen holder.…”