1. Dr. Alvin Yeh Department of Biomedical Engineering Dr. Arne Lekven Department of Biology Josh Bergerson Normangee High School

2. Components of Engineering

3. Faculty Members

4. Lab Group Members

5. Angiogenesis New blood vessel (capillary) formation Important in tissue growth & repair Excessive  cancerInsufficient  stroke

6. Angiogenic Process  ECs detach from wall  Degrade & penetrate basal lamina  Invade surrounding ECM

7. Extracellular Matrix  Scaffold; structural support  Adhesive contact sites  Mechanical & biochemical signals

8. Research Question What effect does changing the collagen fiber stiffness and thickness have on angiogenic patterns in vitro? angiogenesis in diseases vascularzing engineered tissue

9. In Vitro Angiogenesis Model Endothelial cell monolayer Collagen gel preparation Serum–free medium

10. Sandwich Modeling

11. Fiber Thickness Polymerizing collagen at varying temperatures Observe angiogenic patterns in matrix

12. Data Acquisition TPF, SHG & Light microscopy SHG detector TPF detector Ultrafast laser Objective

13. Two-Photon Microscopy  Used to create 3D images from optical sections  Detects sample’s fluorescence (cell/GFP/etc)  Wavelength ~ 405nm TPF-3DTPF-2D

14. Second Harmonic Generation (SHG)  Sample mixes 2 photons  Detects collagen; crystal, repeating structure  Not measuring fluorescence  Wavelength ~ 480 nm Second Harmonic Generation (SHG) E2E2 E1E1 hv in Virtual States SHG TPF+SHG

15. TPF & SHG  Imaging depth ~ 500um  Laser Bandwidth ~ 133nm centered at 800nm  Ability to image living cells  10 femtosecond pulse laser (1x10 -14 s)  PMT detectors

16. Data Analysis  Morphology  Cellular Growth  Lumen Development 1 mg/ml 2.5 mg/ml Brightfield TPF+SHG

17. Possible Classroom Application Physics: Optics Wave properties Mechanics Chemistry: Electromagnetic Spectrum Behavior of Electrons Biochemistry

18. Acknowledgements  TAMU E3 RET Program  National Science Foundation  Nuclear Power Institute  Dr. Alvin Yeh Research Group: Tissue Microscopy Lab  Dr. Arne Lekven Lab Group

19. Conventional Microscopy  Non-laser light source  Snapshot rather than scanning  Imaging depth ~20um

20. Single Photon Microscopy  Imaging depth ~100um  Continuous laser excitation  Pinhole allows for optical sectioning  Photobleaching of fluorescent probes

21. Tracking Gene Expression  Phenotypic expression of brain development genes is know but specifics are not (wnt1, pax2, fgf8)  Genes can be tracked by tagging with fluorescent proteins  Allows detection of gene being “turned on” during development

22.  Grown on collagen or fibrin scaffold Engineered Tissue Scaffolds skinblood vessels tendonligament soft connective tissue

23. Spectral Two-Photon Microscopy  16 PMT spectrometer  Computational linear unmixing

24. Spectral Two-Photon Microscopy  To detect gene expression  Simultaneous detection of multiple fluorescent proteins  Real-time study of live embryonic development

25. Second Harmonic Generation (SHG)  Detects collagen  Requires crystal, repeating structure  Not measuring fluorescence

26. Optical Coherence Microscopy (OCM)  Collects light reflected from sample (morphology)  Collects data via spectral detector (significant power loss)

27. Optical Coherence Microscopy (OCM)  Detects fibrin, collagen, and cell  Factor out collagen and cell through TPF & SHG  Observe growth of cell scaffold under various conditions

29. Wnt1 Gene Expression  Mark Feltner, M.A.  Skyline High School  Dallas, Texas

30.  Laser’s 2-photon emission, in conjunction with fluorescent proteins, is providing us with more detailed imaging of developing embryos.

31.  Several of the genes discussed are oncogenes – they can, under some circumstances, initiate cancerous growth.  (Q: these include which: wnt 1, wnt 8 (all wnt’s??) spt, pax 2…others?)

32.  Each gene is involved in early brain development. The sp5 gene expression, for instance, is involved in development of the midbrain, hindbrain and spinal cord, but not the forebrain. (Holly’s schematic here – circular, yellow. NB p 11. Shows mb/hb/sc/fb regions in yolked embryo)

33. wnt1pax2a fgf8 Multiple genes work together in the same place at the same time

34.  In early zebrafish emryos, the mesoderm and endoderm are initially mixed.  They differentiate.

35. But these are ‘just’ fish, right? How is this relevant to us?  In every vertebrate embryo, there is always a midbrain-hindbrain boundary. This goes for all mammalian species, including us, the mighty Homo sapiens.

36. In fact, this diagram helps illustrate the commonalities of gene expression that all vertebrate species share.

37.  To recap: Before you can actually see a difference in cell formation, the cells are already expressing the various genes that will cause them to differentiate.  The wnt1 gene appears to be conserved across multiple vertebrate species, including humans.  Thus: understand zerbrafish early brain development, and we can better understand mammalian gene expression of brain development.

38. This is huge.  Currently, we know what expression of wnt1 gene does, but nobody knows the mechanism of how this gene gets turned on.  This is one question that we hope to answer. Of course, there are many more.