CHAPTER 6 Illumination 43
results in loss of contrast and therefore information. Especially for live cell imaging
applications you need to protect living cells against phototoxicity and to reduce
photobleaching. The cells also need to be protected from the heat generated
by the light source and from the vibrations caused by mechanical filtering and
switching devices. If you consider this, you will enjoy happy living cells.
LED Light Sources Are a Major Advance for
Fluorescence Microscopy
LEDs are the more up-to-date light source and are becoming more and more
commonplace. LEDs are more expensive and you normally can’t replace them by
yourself. But they are energy efficient: LED arrays have good longevity and are
quite reliable – the average life of an LED bulb is around 50,000 hours. They produce
a brilliant white light with a slight blue tint color temperature or a specific
narrow wavelength peak. They are low in heat emission. You can run an LED bulb
for hours to days and it will not heat up as much as some of the now rarely used
high pressure mercury arc light sources. You may use your microscope equipped
with LED in exactly the same way as with a halogen bulb, but they are especially
useful for fluorescence microscopy. Fluorescence microscopy requires an intense
light source at the specific wavelength that will excite fluorescent dyes and proteins.
LEDs are available
in a variety of colors that match the excitation bandwidth
of many commonly used fluorescent dyes and proteins.
Mercury Arc (HBO) Lamps
Mercury arc lamps – HBO – are still the ideal light sources for applications that
require high intensity spectral lines emitted in the deep UV to visible light regions.
They are traditionally used in fluorescence microscopy. They can generate
ample light at desired wavelengths, but only a small percentage of the projected
light is useful in any particular application. The other wavelengths need to be suppressed
to avoid background noise that reduces image contrast and obscures the
fluorescent light emissions. This process of suppressing extraneous light is complex,
expensive and only partially effective: even after decades of refinements,
the best filters are not 100% percent successful at blocking the bleed through of
non-specific photons. Nevertheless, mercury arc lamp remains a workhorse in
fluorescence microscopy and is still considered one of the best illumination sources,
especially for those fluorophores whose excitation maxima coincide with the
spectral lines emitted by the hot mercury plasma. Mercury arc lamps are tedious
to align within the lamp housing.