Biomedical Engineering Reference
In-Depth Information
and dissociation kinetics (Axelrod
et al.
, 1976; Ellenberg
et al.
, 1997). During
a FRAP measurement, a region within the cell of interest either produces
autofl uorescence or contains proteins that have been tagged with a fl uores-
cent probe (see 'GFP' section) which is photobleached by an intense beam
of excitation light. The time taken for the region to recover a fl uorescence
signal is recorded and can be used to measure diffusion characteristics.
Variations include inverse FRAP (iFRAP), in which the entire cell except
for the region of interest is photobleached (useful for diffusion, and kinet-
ics detection) (Dundr
et al.
, 2002), and fl uorescence loss in photobleaching
where the same area is photobleached repeatedly (useful for determining
compartment connectivity and kinetics) (Ellenberg
et al.
, 1997; Phair and
Misteli, 2000).
FRET
Fluorescence/Forster resonance energy transfer (FRET) provides a mea-
surement of intermolecular interactions (Hoppe
et al.
, 2002), specifi cally
the energy transfer between two adjacent chromophores. In practice, FRET
is used to determine the localization and interaction of a variety of cellu-
lar components over time (Miyawaki
et al.
, 1997), especially fl uorescently
labeled molecules (Sourjik and Berg, 2002).
4.4.6 Cell tracking
When cell-seeded scaffolds are implanted for
in vivo
experimentation, dif-
ferentiating between the seeded cells and host cells can be a diffi cult propo-
sition. The distinction between host and implanted cells must be determined
to establish the extent of the implanted cell's effects. Targeting methods
include sensitive quantum dots which are resistant to host tissue autofl uo-
rescence (Rosen
et al.
, 2007) and iron oxide magnetic nanoparticles (Farrell
et al.
, 2008); fl uorescent tags and Lac Z reporters have been used (Rosen
et al.
, 2007).
4.5 Future trends
Recent advances in biotechniques have been developed to help rapidly
and accurately detect small quantities of molecules of interest. For exam-
ple, real-time PCR was invented to detect an infi nitesimal amount of DNA
through repetitive amplifi cation of the target molecule. Unlike real-time
PCR that allows for accurate detection of a small quantity of DNA, detection
of a low amount of protein still remains challenging. Thus, the immuno-PCR
technology that is capable of amplifying signals of target proteins using the
PCR technology is introduced in this section.
