Molecular Imaging
Alfred Song
National Science Foundation Integrative Graduate Education Research
Traineeship Program
The University of Texas at Austin
The University of Texas MD Anderson Cancer Center
Gelovani’s Group
• Juri Gelovani
– Department of Experimental Diagnostic
Imaging
– Among the first to develop a PET reporter
gene for in vivo molecular imaging
– Previously at Memorial Sloan-Kettering
– Current: In vivo imaging, drug development,
molecular biology
• Research Group
– ~30 people
– Medicine, chemistry, biology, pharmacology,
computer science
www.mdanderson.org
Stains Are Useful for Imaging
Biological Tissues
• Used because tissues, unless
pigmented, are low in
contrast.
• Dyes and vital stains are
colored or fluorescent
chemicals or antibodies that
bind selectively to certain
molecules and increase
contrast.
www.neurostructural.org/services.htm
• Vital stains keep the cell
alive, most others kill the cell.
They are rare.
retina.umh.es/Webvision/VisualCortex.html
Reporter Genes: Another Way to
Increase Contrast
• The reporter gene produces a protein product which is
able to signal where, when, and in what quantity it is
being translated.
• Reporter genes are inherently vital i.e. keeps your cells
alive
• Reporter genes allow to image structure and function.
• Attach a reporter to your gene of interest.
– Fusion Gene is your gene of interest fused to your reporter gene
– Fusion Protein is the expression of your Fusion Gene
Fusion Genes
Fusion
Gene
Gene or Regulatory Region of Interest
Reporter
Transcription
Translation
Reporter
(GFP)
Mouse
Electroporation,
Fusion
Protein
Recombination,
Selection,
Mating,
$30,000.
life.nthu.edu.tw/~labwwc/
staff.science.uva.nl/~zoon/sms/pictures/gfp.jpg
www.mshri.on.ca/nagy/gallery.htm
GFP Driven by the Engrailed
promoter
genetik.fu-berlin.de/institut/en_GFP_fly3.jpg
Commercially Available GFP Fish
www.beverlytang.com/photos/gfp_fish.jpg
Alba the GFP Bunny
bioephemera.com/wp-content/uploads/2007/04/albagreen.jpg
Aequorea victoria
www.conncoll.edu/ccacad/zimmer/GFP-ww/aequorea.jpg
Shimomura O, Johnson F, Saiga Y (1962). "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea". J Cell Comp Physiol 59: 223-39
Fluorescence Reporters Come in
Many Colors
Dunn 2006
Other Reporter Genes
• Bioluminescence reporter genes
– Enzymes from fireflies catalyze a reaction that
produces light.
• PET reporter genes
– Does not emit light, but traps radiopharmaceuticals
inside cells that possess the reporter gene.
– This reporter is also an enzyme. It changes the
polarity of the radiopharmaceutical to trap it inside the
cell.
Bioluminescence Reporter Gene
• Enzyme: Firefly Luciferase
• Substrate: Luciferine
• Luciferase catalyzes the
reaction of luciferin and
ATP into Adenyl-luciferin.
When this molecule
degrades it produces light.
www.britannica.com
www.sigmaaldrich.com/img/assets/4201/Luciferase_Reaction_Scheme.gif
Transgenic Luciferase Tobacco
Plant
•In vivo imaging is
possible.
•Water the plant with a
solution containing
luciferin and it begins to
glow.
www.answers.com/topic/glowing-tobacco-plant-jpg
Luciferase Complimentation
Imaging (LCI)
• A method to image molecular interactions.
• Break luciferase into two pieces, make two
fusion genes.
• If fusion genes interact, luciferase pieces will
interaction and signal.
Luker et al., 2004
Luciferase Complimentation
Imaging (LCI)
• Feed the cells or organism luciferin
• If the cell lights up, you know that the two
proteins interact under the specified
conditions
• If the cell does not light up, your conditions
may not cause interaction
Positron Emissions Tomography
(PET) Reporter Gene
• Enzyme: Herpes simplex virus thymidine kinase
(HSV-TK) is an enzyme that adds phosphates to
thymidine.
• Substrate: Radioactive thymidine analogues
(radiopharmaceuticals) are trapped within the
cell when phosphorylated.
• Radioactivity is imaged in vivo via clinical or
microPET.
Three Dimensional Cell Culture
• Classically cell culture has been on flat plates.
• 3D culture mimics the three dimensional
environment in tissues of the body.
• 3D cell culture yields differing results for stress
experiments than 2D cultures.
– Cells are more resilient to insult
– Possibly due to HSP expression in a hypoxic cores
and/or cell-cell signaling.
3D Culture: Spheroids
Gelovani et al., 2007
3D Culture: Spheroids
• Gelovani has a stream-lined spheroid
culture technique, as well as an arsenal of
reporter genes, and a full time,
experienced molecular biologist (Najjar).
• Plans are in place to begin co-culturing
cells with an endothelial cell line which
they hope will produce a vascular network.
Multiphoton Microscopy (MPM)
• A technique that could combine enhanced
fluorescence imaging, ablation by laser, and
possibly in vivo optical imaging.
• Characteristics of MPM
– Better depth penetration
– Simultaneous imaging of multiple fluorescence
reporter genes
– Cellular and subcellular laser surgery/ablation
• Currently, Gelovani is not able to image through
the other side of the spheroid
Multiphoton Microscopy
www.aecom.yu.edu
www.childrensmrc.org/images/upload/multi1(1).JPG
Lasers: Ti:Sapphire
Pulsed N2
~$150K
100 fs pulse duration
photonics.light.utoronto.ca/img_fac/image025.jpg
Multiphoton Microscopy
Centonze,V.E and J.G.White. (1998) Biophysical J. 75:2015-2024
www.loci.wisc.edu/multiphoton/mp.html
Images of acid fucsin stained monkey kidney taken at a depth of 60 µm by confocal (left) and multiphoton microscopy (right).
Combination 2-photon (red and green) and 3-photon (blue) image of C.elegans embryo
Benefits of Collaboration
• A combination Necrosis/Apoptosis reporter
gene
• In vitro 3D cell culture and in vivo
experiments to investigate the relative role
of necrosis and apoptosis to thermal insult
Conditions for Collaboration
• Development of Necrosis/Apoptosis
reporter gene
• Construction or access to MPM
– Cost: ~$200,000 and a semester to build
– Adela Ben-Yaker (ME dept) is an expert on
ultrafast lasers and has done MPM ablation in
the past
– IGERT facilities have an MPM, but charges a
fee