Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 31

3 Specimen Fixation

Fixation is a chemical process intended to impede autolysis or putrefaction of the
specimen by stopping biochemical reactions, thereby preserving tissues. It also
inactivates bacteria, fungi, and viruses, providing biosafety for lab personnel
(Rubbo et al. 1967 ). Thus, fixation is an important and necessary step in tissue
preparation for microscopy. Certain fixatives, such as glutaraldehyde, are known to
induce specimen fluorescence (Collins and Goldsmith 1981 ). This fixation-induced
fluorescence is caused by a reaction between the amines and proteins from the
tissue and the aldehyde groups in the fixative generating fluorescent products
(Wright Cell Imaging Facility 2013 ). Thus, for fluorescence microscopy, it is
important to select a fixative that will provide adequate preservation of tissue
while inducing minimal fluorescence. The authors have found freshly prepared
paraformaldehyde (4 %) to be a suitable fixative for fluorescence microscopy.

4 Intrinsic Autofluorescence, Fixation-Induced

Fluorescence, and Nonspecific Binding

A certain level of intrinsic fluorescence is expected from tissues due to the presence
of flavins and porphyrins. This phenomenon can be useful for orientation purposes
during microscopic analysis particularly when non-visible dyes, such as Cy5, are
used. However, high levels of background fluorescence can interfere with the
visualization of microorganisms by fluorescence microscopy. As discussed above,
fixatives can also induce autofluorescence in tissues. The emission spectra of
intrinsic or induced autofluorescence is very broad compared to the spectra of
typical fluorophores used for fluorescence microscopy, which makes it difficult to
separate wanted from unwanted fluorescence by traditional filtering methods
(Wright Cell Imaging Facility 2013 ).
Several techniques using quenching chemical solutions have also been devel-
oped with the goal of reducing unwanted tissue fluorescence. These methods
include the use of pontamine skye blue, Sudan black B, sodium borohydrate, trypan
blue, and ammonia-ethanol (Cowen et al. 1985 ; Sun et al. 2011 ; Oliveira
et al. 2010 ; Srivastava et al. 2011 ; Raghavachari et al. 2003 ); however, their
efficacy is variable. Baschong et al. ( 2001 ) studied the effect of three separate
reagents on reducing autofluorescence of three different types of tissue. These
authors found that no single reagent was able to decrease autofluorescence in all
samples and that the selection of an appropriate autofluorescence quenching agent
required a trial-and-error search process.
The nonspecific binding of molecular probes in tissue samples is another prob-
lem that can occur during imaging of biofilms. While designed to be specific
for targeted microorganisms, probes for immunofluorescence and fluorescence in
situ hybridization (FISH) can be subject to significant nonspecific binding.