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Quantitative Image Analysis of Birefringent Biological
Materials - latest research
The liquid crystalline nature of biological materials
makes them ideal candidates for study with the polarised light microscope,
however advances in computer imaging, and microscope controllability have
allowed us to develop a technique that can not only present us with
measurements of retardation but also molecular alignment. This technique
is described briefly here, and in more detail in Quantitative
Image Analysis of Birefringent Biological Material on this site -
the purpose of this article to demonstrate some of our results using this
technique
Brief Description
An adjustment to the standard polarised light
microscope allows quantification of relative retardation and orientation
for weakly birefringent samples , (relative retardation below 50nM - a
typical value for thin sections of many biological materials.). The
standard polarised light microscope set-up consists of a polariser both
above and below the sample. They are crossed- their vibrational axes are
at 90 deg. . As light passes through the first polariser it is split into
two perpendicular rays, that will travel through the sample at different
velocities, the slower wave being retarded relative to the fast wave. This
difference is the relative retardation measured in nm. As the two rays
leave the sample they are out of phase- the second polariser (or analyser)
recombines the rays, where they interfere. For samples with a retardation
of less than 200 nm, a compensator plate is added between the polarisers
to bring this interference within the visible range. This plate is
traditionally set at 45 deg. to the polarisers, however adjusting this
angle by 4.5-7.5 deg. can dramatically increase the colour contrast,
optimised for retardations below 50nM. When combined with the latest image
capture and analysis software , motorised microscope stage and digital
cameras it is possible to accurately evaluate both sample retardations and
orientations, at individual points, or averaged over areas.
The technique itself was discovered by Mae-Wan Ho and
Michael Lawrence whilst examining developing, and freshly hatched fruit
fly larvae (Drosophila melanogaster). In her book
The Rainbow And The Worm, Dr. Ho describes the
living embryo as if it were 'straight out of a dream' - indeed the living
organism viewed in this technique is an amazing sight. She goes on
....As it crawls along, it weaves its head from side
to side flashing jaw muscles in blue and orange stripes on a magenta
background. The segmental muscle bands switch from brilliant turquoise to
bright vermillion, tracking waves of contraction along its body. The
contracting body-wall turns from magenta to purple , through to
irredescent shades of green, orange and yellow. The egg yolk, trapped in
the alimentary canal, shimmers a dull chartreuse as it gurgles back and
forth in the commotion. A pair of pale orange tracheal tracts run from
just behind the head down the sides terminating in yellow spiracles at the
posterior extremity.
Interestingly enough all organisms examined were
polarized along the anterior-posterior axis, the colours of the different
tissues of the body are at a maximum when the axis is appropriately
aligned in the optical system- this is demonstrated by the drosophila
larvae in fig 2a, as it curls around. circle
Of course the (almost) non-invasive study of living
organisms can provide significant information with regard molecular
organisation, however to study a sample quantitively it must be examined
in a number of orientations, whilst keeping still - something living
dosophila larvae don't do! continue>>
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