1. Non-linear Optical Microscopy: Viewing embryonic development of zebra fish Esther Johnson Del Rio High School – Physics Dr. Alvin Yeh, Associate Professor of Biomedical Engineering Dr. Arne Lekven, Associate Professor of Biology

2. Dr. Yeh’s Research Group Dr. Alvin Yeh - Associate Professor Biomedical Engineering Yuqiang Bai – Engineered tissue using integration of optical coherence Po-Feng Lee – Imaging angiogenesis with nonlinear optical microscopy Chao Wang – Using two-photon microscopy as compared to confocal fluorescence microscopy

3. Holly Gibbs

4. Dr. Arne Lekven Associate Professor of Developmental Biology

5. So what is the objective? Can we develop the instrumentation for nonlinear optical microscopy (NLOM) using broadband, ultra-short pulses to improve the longitudinal study of engineered tissues and model organisms. Can we image more fluorescent proteins at once by combining NLOM with spectral (16 channel) detection?

6. Why imaging? “Most systems biology approaches involve determining the structure of biological circuits using genomewide “-omic” analyses. Yet imaging offers the unique advantage of watching biological circuits function over time at single- cell resolution in the intact animal.” Megason, Sean and Fraser, Scott “Imaging in Systems Biology” Cell 130. 9/7/2007. pp784-795

7. Potential Real World Applications Dr. Yeh’s & Dr. Lekven’s Research Groups: Looking for better ways to connect the world of molecular biology with the properties and functions of various tissues and organs Working to unlock the mechanisms of embryonic development with potential applications in stem cell replacement therapy, cancer research, and other biomedical arenas

8. WHAT IS NON-LINEAR OPTICAL MICROSCOPY????? Noninvasive Excite target cells Great detail Produce 3D images Setup SHG TPF OVERLAY 2D images3D stack image

9. Galvanometer driven mirror SHG detector TPF detector Ultrafast laser Objective Dr. Yeh’s Research Group Laser

11. Two photon microscopy Two photons both excite and detect specific gene patterns 350 500 650 350 500 650 Wavelength (nm) Intensity (a.u.) Y Y Y Y nt mh b One-Photon Excited Fluorescence (OPEF) Two-Photon Excited Fluorescence (TPEF) 1 photon 2 photon v.

12. Why zebrafish? Transparent Easy to observe Simple organisms Share many common vertebrate developmental features

13. ..\..\Zebrafish-development.mov

15. “Making babies”

16. In situ hybridization

17. Our target sequence was ECR-20 (an evolutionarily conserved region just before the wnt 1 gene).

18. Creating a transgenic fish Genetic probes encoded within DNA Benefit: can be observed over a period of time Potential Problem: trans- genes can be difficult to induce

20. Linear Unmixing Linear unmixing Measurement is a linear sum of constituent spectra + B x + C x AF ref eGFP ref citrine ref = fit sse=Σ(measurement-fit) 2 measurement A x

21. Summary Zebrafish provide a developmental model. Noninvasive method 3D image NLOM can excite multiple fluorescent proteins at the same time.

22. Potential Project Ideas Exploring how lasers can be used in microscopes with lenses Separating various color components utilizing spectroscopy

23. Acknowledgements TAMU E3 Program National Science Foundation Nuclear Power Institute Texas Workforce Commission Holly Gibbs Dr. Arne Lekven Dr. Alvin Yeh Kirsten Brink