Making Light Work in Microscopy
Professor Tony Wilson
Department of Engineering Science, Oxford
3:30 pm Monday, 9 February 2009, EN101
(Ground Floor, EN Building), Hawthorn.
There are many factors
that influence the final design of a confocal microscope. Among
these are the choice of light source and the method of achieving
optical sectioning or confocal operation. The choice of illumination
has traditionally been restricted to available laser lines, however
the recent introduction of white light supercontinuum (WLS) sources
has provided much greater freedom and, in combination with spectral
detection, greater versatility in confocal fluorescence microscopy.
We will demonstrate the use of a WLS source in a reflection mode
confocal microscope together with spectral detection to provide
sprectralluy resolved images in three-dimensions, This approach
has enabled the imaging of specimen features that are not apparent
in either laser illuminated confocal or conventional microscopy.
Microscopic imaging of thick biological specimens
is often detrimentally affected by specimen-induced aberrations
that cause a loss in signal strength together with reduced resolution.
Aberrations can be corrected using adaptive optics, where a deformable
mirror introduces equal but opposite aberrations into the optical
path . We will discuss the design of a wave front sensorless adaptive
system for conventional fluorescence microscopy and optical sectioning
microscopy using a structured illumination approach.
Whichever light source and method of obtaining
confocality is chosen it is still necessary to focus the microscope.
This operation is the bottleneck to high-speed image acquisition
since it usually requires a mechanical system to physically move
the specimen so that the region of interest is brought into the
focal region of the objective lens. In scanning microscopes it is
also necessary to introduce a scanning mechanism so that the focal
spot is scanned across the focal plane of the specimen. It is straightforward
to introduce this scan optically using, for example, galvanometer
mirrors since the objective lenses are usually designed according
to the sine condition. It is clearly be desirable to develop an
optical method of focussing which would leave the specimen stationary.
We will describe such a system where the refocusing may be achieved
remotely from the specimen at high speed without introducing spherical
aberration that is common to other optical refocusing systems. In
this way can position the focal spot at an arbitrary point in the
focal region of a high-aperture microscope objective at high speed.
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