5. Various Imaging Techniques
• Magnetic Resonance Imaging (MRI)
• Positron Emission Tomography (PET)
• Single-Photon Emission Computed Tomography (SPECT)
• Fluorescence Resonance Energy Transfer (FRET)
• Fluorescence
• Bioluminescence
6. Magnetic Resonance Imaging (MRI)
• Used to visualize the inside of living organisms
• Demonstrates pathological or other physiological alterations of living
tissues (i.e. tumors)
• Uses radio frequency signals to acquire images
• Based on the relaxation properties of excited Hydrogen nuclei in
water
http://en.wikipedia.org/wiki/Image:User-FastFission-brain.gifhttp://en.wikipedia.org/wiki/Image:3Dbrain.gif
7. Positron Emission Tomography (PET)
• A nuclear medicine imaging technique
that produces a 3D image or map of
functional processes in the body
• Uses a short-lived radioactive tracer
isotope which decays by emitting a
positron (has been chemically
incorporated into a metabolically
active molecule) and is injected into
the living animal, usually in the blood
• Commonly used alongside CT scans
or MRI scans, giving both anatomic
and metabolic information
http://en.wikipedia.org/wiki/Image:PET-MIPS-anim.gif
8. Single-Photon Emission Computed
Tomography (SPECT)
• A nuclear medicine tomographic imaging technique using gamma rays
able to provide true 3D information
– A 2D view of the 3D distribution of a radionucleotide from
multiple angles
• A computer is used to apply a tomographic reconstruction algorithm to
yield a 3D dataset
– Can be manipulated to show thin slices along any chosen axis of
the body
9. Fluorescence Resonance Energy Transfer (FRET)
• Energy transfer mechanism between two fluorescent molecules
• Useful tool to quantify molecular dynamics in biophysics, such as protein-
protein interactions, protein-DNA interactions, and protein conformational
changes
– Monitors the complex formation between two molecules, one is labeled
with a donor and the other with an acceptor, which are then mixed
– When they dissociate, the donor emission is detected upon the donor
excitation, but when together, the acceptor emission is predominant
10. Fluorescence
• Production and emission of light by a living organism as the result of a
chemical reaction during which chemical energy is converted to light
energy
– Uses an external light source with a low-pass filter to excite the fluorescent
molecules
• Green Fluorescent Protein, originally found in the Aequorea victoria
species of jelly fish
– Been biochemically modified to produce Green, Yellow, Blue, Cyan, and Red
Fluorescent Proteins for use in various research techniques using a reporter
• Limited by tissue autofluorescence, as well as the light being able to
first get into the living model and sensing the target fluorescent
molecule, then having that fluorescence get back out of the model and
to the detector (a lot of scattering occurs)
http://wwwchem.leidenuniv.nl/metprot/armand/images/029l.jpghttp://en.wikipedia.org/wiki/Image:Aequorea_victoria.jpg http://www.upenn.edu/pennnews/photos/704/mice.jpg
11. Bioluminescence
• Bioluminescence is the production and emission of light by a
living organism.
• Bioluminescence occurs widely in marine vertebrates
and invertebrates, as well as in some fungi, microorganisms
and terrestrial invertebrates.
• Some symbiotic organisms carried within larger organisms
produce light
Cherry et al. 2004
12. Autofluorescence
Cells contain molecules, which become fluorescent
when excited by UV/Visual radiation of suitable
wavelength.
This fluorescence emission, arising from endogenous
fluorophores, is an intrinsic property of cells and is
called auto-fluorescence which is different from
fluorescent signals obtained by adding exogenous
markers.
rm
15. Goals of in vivo AFPs measurements
• Measuring molecular distances
• Detecting conformational
changes
• Detecting interactions
• Localizing interactions
• Following interaction dynamics
• Reporting enzymatic activities
and intracellular conditions
16. Tracking Messengers
Small molecules as second messengers play a central role in signal transduction
.
One such ubiquitous messenger is nitric oxide (NO), which is involved in
various physiological and pathophysiological processes.
Diaminofluoresceins and diaminocyanines have been described as small
molecule-based sensors for the detection of NO.
However, these molecules do not sense NO directly but react with oxidized NO
products to yield a highly fluorescent product.
18. • A fluorescent sensor that allows for the first time a direct detection of NO
was recently introduced by Lippard and colleagues.
•
Fluorescein-based sensor (CuFL) for detecting NO
www.acschemicalbiology.org/VOL.2 NO.1 • 31–38 • 2007
19. VOL.2 NO.1 • 31–38 • 2007
Creating Fluorescent Sensors for Enzymatic Activities by Design
• Fluorescein derivatives (dubbed TokyoGreens) (12) as sensors for β-galacto
sidase activity.
www.acschemicalbiology.org/VOL.2 NO.1 • 31–38 • 2007
20. • Rhodamine-based probes as sensors for esterase activity.
Esterases release the phenol of the so-called trimethyl lock group , and this
leads to rapid lactonization and liberation of the fluorophore
www.acschemicalbiology.org/VOL.2 NO.1 • 31–38 • 2007
21. Measuring Ions
• Zinc, although considered a trace element, is an abundant metal
ion whose concentration within eukaryotic cells is 100 µM.
• Zinc is bound to various proteins, including transcription factors,
and acts as a cofactor in several enzymes.
• The total concentration of zinc in cells is relatively high, whereas
the concentration of free or rapidly exchangeable zinc is very low.
• Estimates of the concentration of free zinc in prokaryotic cells are
in the femtomolar range.
22. • Measuring free or rapidly exchangeable zinc at picomolar or lower
concentrations in the presence of high concentrations of calcium
and magnesium thus requires a highly specific and sensitive
fluorescent sensor.
24. AFPs as sensors for biological processes face limitations.
First, AFPs are relatively bulky, in the best case monomers of 240 residues,
and size matters for all applications for which the distance between the AFP
and the activity to be recorded is critical.
Second, the spectral range of AFPs is limited. For example, no useful AFPs
are available in the near-infrared region, and different pairs of AFPs for
simultaneous FRET measurements in living cells have not yet been
established.
Third, for the construction of sensors for various crucial biomolecules and
enzymatic activities, AFPs offer no obvious solution.
Future outlook
