42 PART II A Deeper Look into a Light Microscope
Let There Be Light – A Glance at
Microscope Light Sources
The variety of available light sources is as rich and overwhelming as deciding
on a type of pasta. There is no best or worst. It also applies that the application
and the type of result that you want determines your light source. In transmitted
light microscopy, either brightfield or phase contrast, both halogen bulbs or LED
arrays
are widely used. Both have advantages and limitations – it’s a matter of preference.
And money …
Stay Traditional with Halogen
Halogen bulbs are the traditional light source with a tungsten filament inside
a glass casing.
The casing is filled with halogen gas such as iodine or bromine,
simply to increase the life span of the filament. They are widely used in transmitted
light microscopes for brightfield and phase contrast, and may even be
bright enough for most applications in polarized light. They are a good choice
for examination
of stained cells and tissue sections, as well as numerous reflected
light applications. Halogen lamps are relatively inexpensive, easy and cheap
to replace. Being full spectrum lamps, halogen lights give off great color integrity,
which basically means
that the sample is going to show up as a true-to-color
life image. Due to their warmer color tint many pathologists for instance use halogen
bulbs as the samples appear to be richer in color. For true to daylight color
impression the halogen light source is combined with a blue conversion filter.
There must be a »but«, for sure. Most of the energy emitted by these lamps is
dissipated
as heat. Over their life span halogen bulb glass starts to darken and
intensity drops down.
Fluorescence Microscopy Is Sensitive in its Favorite
Light Source
The most basic requirement of a fluorescence microscopy light source is closely
matching the excitation wavelength of the fluorochrome to achieve a high-contrast
image – that is, an image with a high signal-to-noise ratio. Wavelengths
that match the fluorochrome strengthen the signal, but any peripheral wavelengths
produce background noise that can overshadow the signal emitted by
the object of interest. Another related requirement is the illumination intensity,
i.e. the number of photons specific to the excitation wavelength that reach the
specimen. Lower intensity is necessary to protect against photobleaching, which