Page 85 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 61

Generally, chemical fixation of cells is the most suitable sample
preparation method for investigation with FTIR. In the past, how-
ever, chemical fixation has been avoided due to the potential for
interference of the fixative with the IR spectrum. In this chapter, we
discuss the influence of chemical fixatives on the FTIR spectrum of
fixed single cells and show FTIR maps that illustrate the differences
in biomolecular localizations in fixed versus unfixed cells. Our discus-
sion of fixation also extends to resected tissues, where we provide a
summary of the different methods employed to prepare these spec-
imens for spectroscopic analysis, together with a review of the diag-
nostic information that can be obtained as a result of these preparations.
In addition to discussing fixed material, this chapter also reports
on recent studies using live cells for FTIR and Raman studies, detail-
ing the quality of spectral information obtained from these experi-
ments, as well as the technical challenges imposed by maintaining
living cells during analysis.
Another fundamental aspect of sample preparation that can
influence cellular biochemistry is the surface on which they are
grown. The surface can induce changes in cell adhesion and motil-
ity, in their proliferation and differentiation and in gene expression. It
is desirable for in vitro cultures to mimic the in vivo environment as
closely as possible and in this context, progress has recently been
made in modelling cellular systems in two-dimensional cultures.
Studies have also been carried out detailing the use of biomaterial
surfaces (Matrigel TM , fibronectin, laminin, gelatin) for this type of
cell culture. The influence of these surfaces on cell morphology
and the spectral information obtained is also discussed.

3.2 Tissue Preparation

3.2.1 Archived Tissue: Paraffin Embedded and
Frozen Specimens
Surgically excised tissue may undergo one of two commonly used
methods of preservation for long-term storage, paraffin embedding,
or flash-freezing. The choice between these two methods is based on
the specific purpose of the resected tissue. Currently, paraffin embed-
ding is the preferred source for the histological examination of tissue
sections by light microscopy. This method involves immersing tis-
sue into a primary fixative, which is usually an aqueous formalin-
based solution. Hydrated formalin (methylene glycol, OH-CH-OH)
is a coagulative protein fixative, cross-linking the primary and sec-
10
ondary amine groups of proteins but preserves some lipids by react-
ing with the double bonds of unsaturated hydrocarbon chains. 11
Following formalin fixation, the tissue is dehydrated through con-
secutive immersions in increasing concentrations of ethanol solution.