Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 38

sugar phosphates. Because of the neutral back bone, diffusion of PNA probes
through hydrophobic cell walls may be facilitated and help overcome the problem
of probe penetration into Gram-negative bacteria. Fazli et al. ( 2011 ) applied
specific and universal bacterial PNA-FISH probes to chronic venous leg ulcer
biopsies to identify wounds containing either P. aeruginosa or S. aureus . The two
groups were then evaluated for the presence of neutrophils. Overall, microorgan-
isms were observed in large aggregates (biofilms) and ulcers harboring
P. aeruginosa correlated with the presence of higher neutrophil counts. This
association between P. aeruginosa biofilms and neutrophils was hypothesized to
cause the persistent inflammatory response and delayed wound healing in
P. aeruginosa -infected wounds. The association of P. aeruginosa and neutrophils
was also observed in experimentally infected mouse wounds using PNA-FISH to
detect P. aeruginosa and DAPI (4 0 ,6-diamidino-2-phenylindole) to detect neutro-
phils (Trøstrup et al. 2013 ). Regardless of the PNA-FISH approach, these examples
underscore the potential for the analysis of spatial relationships between microor-
ganisms and host cells to aid in the understanding and diagnosis of disease.
8 Microscopy
The use of microscopy to demonstrate the presence of biofilms in tissue samples has
been increasing. Akiyama et al. ( 1996 ) used light and transmission electron micros-
copy of skin biopsy thin sections to image the time course of S. aureus growth
within inoculated mouse wounds. Microcolonies of S. aureus were detected as soon
as 3 h after inoculation. James et al. ( 2008 ) evaluated samples from chronic and
acute human wounds using Gram staining and light microscopy as well as scanning
electron microscopy (SEM) and found that chronic wounds were more likely to
harbor biofilms. Freeman et al. ( 2009 ) also used Gram staining and light micros-
copy to demonstrate the presence of biofilms in horse wound biopsies.
While light microscopy has been successfully used to detect bacteria in tissue,
fluorescence microscopy has been fundamental for the study of biofilms. Not only
does fluorescence microscopy provide excellent spatial resolution and the detection
of broad emission profiles, it also enables the labeling of specific tissue structures
and bacterial cells within a sample (Coling and Kachar 2001 ). In particular, the
application of confocal scanning laser microscopy (CSLM) has revolutionized
biofilm imaging. Conventional microscopy techniques require that samples are
observed as thin sections on a slide but CSLM allows the examination of fully
hydrated relatively thick biofilms. This enables sample preparation with minimal
manipulation, helping to ensure that biofilms keep their original morphology and
architecture. This attribute is particularly valuable for examining spatial relation-
ships between bacteria within biofilms and host cells and tissue. Confocal micros-
copy was developed and patented by Marvin Minsky in 1955. It was primarily used
in physical and medical sciences until the 1990s when it was first applied for the
study of biofilms (Lawrence et al. 1991 ). CSLM uses optical imaging to create a