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Carl Zeiss Microscopy Online
Campus
Fundamentals of
Light-Emitting Diodes (LEDs)
Introduction
Among the most
promising of emerging technologies for
illumination in optical
microscopy is
the light-emitting diode
(
LED
). These versatile
semiconductor devices
possess all of
the desirable features that incandescent (tungsten
halogen) and arc
lamps lack, and are
now efficient enough to be powered by low-voltage
batteries or
relatively inexpensive
switchable power supplies. The diverse spectral
output afforded
by LEDs makes it
possible to select an individual diode light
source to supply the
optimum excitation
wavelength band for fluorophores spanning the
ultraviolet, visible,
and near-infrared
regions. Furthermore, newer high-power LEDs
generate sufficient
intensity to
provide a useful illumination source for a wide
spectrum of applications in
fluorescence microscopy (see Table 1),
including the examination of fixed cells and
tissues, as well as live-cell imaging
coupled to F?
rster resonance energy
transfer (
FRET
)
and lifetime measurement
(
FLIM
) techniques. The full
width at half maximum (
FWHM
;
bandwidth) of a typical quasi-
monochromatic LED varies between 20 and 70
nanometers (see Figure 1), which is
similar in size to the excitation bandwidth of
many
synthetic fluorophores and
fluorescent proteins. As compiled in Table 1, LEDs
having
output wavelengths in the
400-465 nanometer range exhibit power levels
exceeding 20
milliwatts/cm
2
,
whereas most of the longer wavelength-emitting
LEDs (green through red)
have power
outputs of less than 10
milliwatts/cm
2
. The broad
spectral profile of several
LEDs in the
535 to 585 nanometer region is due to the fact
that these diodes incorporate
a
secondary phosphor that is excited by a violet or
ultraviolet primary LED, thus reducing
power output and broadening the
spectral profile. Thus, the green to yellow-orange
excitation region, one of the most
useful for common fluorophores such as TRITC,
MitoTrackers, and orange or red
fluorescent proteins, remains a downside for those
applications (such as FRAP and
photoactivation) that require high light
levels.