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Fluorescence illumination and observation is the most rapidly expanding microscopy technique employed today, both in the medical and biological sciences, a fact which has spurred the development of more sophisticated microscopes and numerous fluorescence accessories.
Introductory
Concepts
Fluorescence is a member of the ubiquitous luminescence family of processes
in which susceptible molecules emit light from electronically excited
states created by either a physical (for example, absorption of light),
mechanical (friction), or chemical mechanism. Generation of luminescence
through excitation of a molecule by ultraviolet or visible light photons
is a phenomenon termed photoluminescence, which is formally divided into
two categories, fluorescence and phosphorescence, depending upon the electronic
configuration of the excited state and the emission pathway. Fluorescence
is the property of some atoms and molecules to absorb light at a particular
wavelength and to subsequently emit light of longer wavelength after a
brief interval, termed the fluorescence lifetime. The process of phosphorescence
occurs in a manner similar to fluorescence, but with a much longer excited
state lifetime.
Fundamental
Aspects of Fluorescence Microscopy
The
modern fluorescence microscope combines the power of high performance
optical components with computerized control of the instrument and digital
image acquisition to achieve a level of sophistication that far exceeds
that of simple observation by the human eye. Microscopy now depends heavily
on electronic imaging to rapidly acquire information at low light levels
or at visually undetectable wavelengths. These technical improvements
are not mere window dressing, but are essential components of the light
microscope as a system.
Anatomy
of the Fluorescence Microscope
In contrast
to other modes of optical microscopy that are based on macroscopic specimen
features, such as phase gradients, light absorption, and birefringence,
fluorescence microscopy is capable of imaging the distribution of a single
molecular species based solely on the properties of fluorescence emission.
Thus, using fluorescence microscopy, the precise location of intracellular
components labeled with specific fluorophores can be monitored, as well
as their associated diffusion coefficients, transport characteristics,
and interactions with other biomolecules. In addition, the dramatic response
in fluorescence to localized environmental variables enables the investigation
of pH, viscosity, refractive index, ionic concentrations, membrane potential,
and solvent polarity in living cells and tissues.
Practical
Aspects of Fluorescence Filter Combinations
Microscope
manufacturers provide proprietary filter combinations (often referred
to as cubes or blocks) that contain a combination of dichroic mirrors
and filters capable of exciting fluorescent chromophores and diverting
the resulting secondary fluorescence to the eyepieces or camera tube.
A wide spectrum of filter cubes is available from most major manufacturers,
which now produce filter sets capable of imaging most of the common fluorophores
in use today.
Light
Sources
In order
to generate enough excitation light intensity to furnish secondary fluorescence
emission capable of detection, powerful light sources were needed. These
were usually either mercury or xenon arc (burner) lamps, which produce
high-intensity illumination powerful enough to image faintly visible fluorescence
specimens. With the introduction of precisExcite™ this has now all changed,
where instead of high intensity arc lamps the illumination is now provided
by high intensity LED /Light Emitting Diodes supplying light fluxes of
similar intensities without the any of the drawbacks of conventional arc
systems.
Focusing
and Alignment of Arc Lamps
Mercury and xenon arc lamps are now widely utilized as illumination sources
for a large number of investigations in widefield fluorescence microscopy.
These lamps all require aligning and focusing the arc lamp in a Mercury
or Xenon Burner with the Microscope, but with the advent of precisExcite™
this has now been made a thing of the past as zero alignment is required
for the 10,000 hour lifetime of the LED source.
Fluorescence
Photomicrography
Photomicrography
under fluorescence illumination conditions presents a unique set of circumstances
posing special problems for the microscopist. Exposure times are often
exceedingly long, the specimen's fluorescence may fade during exposure,
and totally black backgrounds often inadvertently signal light meters
to suggest overexposure. Added to this, existing bulb technology further
complicates the results achieved as the light output of the bulb degrades
very quickly and therefore the microscopist has to determine whether it
is bulb degradation or the specimen's fluorescence degradation that he
is measuring. The precisExcite™ changes all of this as the light output
degradation is measured over 1000's of hours of continuous use and not
10's of hours of use. Using this light source, microscopists will be better
able to characterize their results as they will not have to correct for
bulb intensity degradation in their findings.
As COOLLED helps to further develop this emerging new market, there are going to be applications that as yet have not been thought of, and therefore if you have any questions, please contact us.
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