Graeme and Nils have been working with Alexander McVey, Till
Bachmann and Jason Crain at the University of Edinburgh's Centre
for Collaborative Optical Spectroscopy, Micromanipulation and
Imaging (COSMIC) on an investigation of Coherent Anti-Stokes Raman
Scattering (CARS) applied to the study of bacterial biofilms. The
experimental apparatus includes two of our ultrafast Sprite Ti:S
lasers. M Squared lasers is the industrial sponsor of the research
and the provision of the hands-free Sprite laser system improves
the usability of the experimental apparatus in addition to
enhancing the stability and simplicity of the method.
In the big picture this is a feasibility study into the use of
CARS to image bacteria in the hope of investigating the effect of
biofilm formation inside the bacteria on their resistance to
antimicrobial drugs.
What is a biofilm?
Crudely put: slime. You might say that forming a biofilm is a
mode of behaviour and growth for a bacterium: for example, in
response to exposure to a particular sort of suitable surface, or
to an attacking agent such as a low dose of antibiotics, certain
bacteria change their behaviour and start to adhere to each other
and / or to a surface (for example, the epithelium in your sinus).
The formation of biofilms on surfaces clearly has major
implications for hospitals and industry. It is known that
legionella bacteria form biofilms which protect them against
disinfectants in industrial settings like cooling towers and
air-conditioning plant. People are then exposed to the contagion
when they use the water or breath the conditioned air.
What is CARS?
Coherent Anti-Stokes Raman Scattering is a microscopy technique
for imaging very small, very complex stuff such as bacterial cells.
The benefits of CARS include that it does this in a non-destructive
way (other methods of imaging living material often have the side
effect of killing or destroying the material being viewed) and that
it can view deeper into a sample - showing more than just what is
on the surface.
Ultrafast, femtosecond lasers are used to produce three electric
fields - the pump, the Stokes and the probe - which are mixed
temporally and spatially and focused on the sample to be imaged.
When the beat frequency (pump minus Stokes frequency) matches a
Raman active vibrational mode, molecules are coherently driven with
the excitation field. (Raman scattering happens when photons are
emitted because of a change in energy levels within the targeted
molecule). This leads to the generation of a strong
anti-Stokes signal which is blue-shifted (higher frequency) from
the incident beam and can therefore be isolated via a dichroic
mirror.
Has it worked?
Yes. So far the system has successfully imaged polystyrene
beads of 2μm (3a-c) and 1μm (3d-f) diameters and E. coli
strain MG1655. The images above are from this preliminary
study.