Biomedical imaging is a key tool for the study of host-pathogen interactions. New techniques
are enhancing the quality and flexibility of imaging systems, particularly as a result of developments
in laser technologies. This work applies the combination of two advanced laser imaging
methods to study the interactions between a virus and the host cells it infects.
The first part of this work describes the theory and experimental implementation of coherent
anti-Stokes Raman scattering microscopy. This technique—first demonstrated in its current
form in 1999—permits the imaging of microscopic samples without the need for fluorescent
labelling. Chemical contrast in images arises from the excitation of specific vibrations in the
sample molecules themselves. A laser scanning microscope system was set up, based on an
excitation source consisting of two titanium-sapphire lasers synchronized with a commercial
phase-locked loop system. A custom-built microscope was constructed to provide optimal
imaging performance, high detection sensitivity and straightforward adaptation to the specific
requirements of biomedical experiments. The system was fully characterized to determine its
performance.
The second part of this work demonstrates the application of this microscope platform in
virology. The microscope was configured to combine two nonlinear imaging modalities: coherent
anti-Stokes Raman scattering and two-photon excitation. Mouse fibroblast cells were
infected with a genetically modified cytomegalovirus. The modification causes the host cell
to express the green fluorescent protein upon infection. The host cell morphology and lipid
droplet distribution were recorded by imaging with coherent anti-Stokes Raman scattering,
whilst the infection was monitored by imaging the viral protein expression with two-photon
excitation. The cytopathic effects typical of cytomegalovirus infection were observed, including
expansion of the nucleus, rounding of the cell shape, and the appearance of intracellular
viral inclusions. In some cases these effects were accompanied by dense accumulations of lipid
droplets at the nuclear periphery. Imaging was performed both with fixed cells and living. It
was demonstrated that the lipid droplets in a single live cell could be imaged over a period of
7 hours without causing noticeable laser-induced damage. The system is shown to be a flexible
and powerful tool for the investigation of virus replication and its effects on the host cell