FRET (Fluorescence Resonance Energy Transfer) and Illumination
FRET (Fluorescence or Förster Resonance Energy Transfer) is a quantum
mechanical process involving the non-radiative transfer of energy
between fluorophores over a small distance (1-10 nm). If the molecules
are close enough, the donor fluorophore transfers its energy to the
acceptor fluorophore. Monitoring the ratio of the fluorophore emissions
provides valuable information to researchers about the spatial
localization of the proteins being studied.
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Successful FRET requires a uniform and stable light source for
accurate fluorescence quantitative data. Read about how a light source
can skew FRET data and how the X-Cite® exacte can generate reliable and repeatable FRET data in ”The importance of a stable fluorescence light source in FRET measurements”.
For those interested in LED technology for FRET experiments, studies have shown how the X-Cite 120LEDBoost
can successfully generate FRET data by providing a uniform field of
illumination, fine intensity control and incredible stability. Read
about “Successful FRET Measurements Using White Light Solid-State
Successful FRET Microscopy Measurements Using White Light Solid-State Technology
Wide field microscopy is the most commonly used
fluorescence microscopy technique. The colorful range of available
fluorescence probes can be imaged with a widefield microscope equipped
with a mercury or xenon arc lamp using a correct combination of
excitation, emission and dichroic filters. For quantitative fluorescence
measurements, such as FRET, critical alignments of a traditional arc
lamp are required to ensure an even illumination field; optical
stability is unreliable due to the nature of these lamps, and a series
of neutral density filters are typically used to tune the light
intensity to a desired level, especially critical for live cell imaging.
All these traditional requirements can now be phased out with new
LED-based systems. Here, we present FRET measurement results using the X-Cite 120LEDBoost system.
With X-Cite exacte, you won’t FRET about your fluorescence light source!
X-Cite customers have found that using the X-Cite exacte system reduces the need to normalize the ratio of the
fluorescence signals. The X-Cite exacte system
provides fluorescence illumination that is more stable than the
traditional arc-lamp and offers greater control over variables which
cause undesirable artifacts in a FRET set up. Researchers can modulate
the light output to optimize imaging conditions and minimize
photobleaching, while having a stable fluorescence light source for
imaging over long periods of time (10-24 hours time-lapse FRET
The X-Cite exacte system
provides unparalleled stability through its DC powered lamp and
Closed-Loop Feedback mechanism, ensuring stable fluorescence
illumination over the course of the experiment – whether it lasts for
milliseconds or days. The Intelli-Lamp® technology ensures worry-free
operation with pre-aligned, long-life lamps.
The following article was published in Bioscience Technology, June 2010 issue, Pg 11.