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.

• Vital stains keep the cell alive, most others kill the cell. They are rare.

www.neurostructural.org/services.htm

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 GenesGene or Regulatory Region of Interest ReporterFusion

Gene

life.nthu.edu.tw/~labwwc/ staff.science.uva.nl/~zoon/sms/pictures/gfp.jpg

Fusion Protein

Transcription

Translation

www.mshri.on.ca/nagy/gallery.htm

Reporter (GFP) Mouse

Electroporation,

Recombination,

Selection,

Mating,

$30,000.

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.comwww.sigmaaldrich.com/img/assets/4201/Luciferase_Reaction_Scheme.gif

Transgenic Luciferase Tobacco Plant

www.answers.com/topic/glowing-tobacco-plant-jpg

•In vivo imaging is possible.

•Water the plant with a solution containing luciferin and it begins to glow.

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

• 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.

3D Culture: Spheroids

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.eduwww.childrensmrc.org/images/upload/multi1(1).JPG

photonics.light.utoronto.ca/img_fac/image025.jpg

Lasers: Ti:Sapphire

Pulsed N2

~$150K

100 fs pulse duration

Multiphoton Microscopy

www.loci.wisc.edu/multiphoton/mp.html Combination 2-photon (red and green) and 3-photon (blue) image of C.elegans embryo

Centonze,V.E and J.G.White. (1998) Biophysical J. 75:2015-2024 Images of acid fucsin stained monkey kidney taken at a depth of 60 µm by confocal (left) and multiphoton microscopy (right).

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