Biology Reference
In-Depth Information
increased risk of shattering. Whilst the imaging system may claim to have a broad
spectral range (e.g. 300-900 nm), the quantum efficiency will only be high over a
limited range. Many of the 'bioimaging' CCDs have been modified so that the quan-
tum efficiency is enhanced at the shorter wavelengths (blue/green). CCDs are tradi-
tionally most sensitive in the far-red/IR range.
Current high-resolution cameras will have a pixel size in the region of
6.45
m. While high resolution is important, there is always a balance that
must be achieved between resolution and sensitivity. For high-quality imaging of
microbial cells, we do not recommend pixel sizes greater than 9
6.45
m
m
m.
6.2
Objectives
Under most circumstances, bacterial cells can be visualised with a 100
objective.
These are available in various apertures and which one to choose is dependent on the
intended purpose. A higher numerical aperture (NA) will result in a trade-off with an
increased amount of light (i.e. sensitivity) but decreased depth of field. Our current
system utilises a 100
NA 1.4 objective. Most standard 100
objectives have an
NA of 1.3 that is suitable for many applications.
To increase the magnification of the image, an Optivar lens (for Zeiss micro-
scopes) can be used. While it will increase the size of the image without loss of
resolution, the placing of the lens between the sample and the objectives will result
in a significantly diminished signal to the CCD.
6.3
Filters
Filters for fluorescence consist of an excitation filter, a dichroic beam-splitter mirror
and an emission filter. These filters are often bought as a set and are housed within a
filter cube. Specialist filter suppliers include Omega Optical and Chroma Technol-
ogy. It is essential to use the most appropriate, and optimal, filter set for the fluores-
cent protein or stain that is being used. Each protein has a specific excitation and
emission wavelength (see
Table 4.2
). Both excitation and emission filters can let
a narrow or wide range of wavelengths through. An emission filter with a wider
bandwidth will be more sensitive but less specific. This can be an issue if dual fluor-
ophores are being imaged in a single sample when fluorescence from one protein can
be observed in the emission channel of the partner protein (crossover).
In order to visualise multiple fluorophores using dual or triple bandpass filter sets
(e.g. GFP/mCherry), a filter wheel is recommended. The excitation filters are
removed from the filter cube and placed in the filter wheel that can then be controlled
to visualise the appropriate signal. When assembling a system, it is important that
filter wheel and camera shutters have the appropriate drivers so that imaging can
be performed rapidly and simply with a single click.
Neutral density filters (grey filters) reduce the transmission of light at all wave-
lengths by an equal amount. They are often used for samples that have unusually high
fluorescence intensities (rare in bacteria) and to protect
live samples from
