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

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

Holly Gibbs

Dr. Arne Lekven Associate Professor of Developmental Biology

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?

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 /7/2007. pp

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

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

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

Two photon microscopy Two photons both excite and detect specific gene patterns 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.

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

..\..\Zebrafish-development.mov

“Making babies”

In situ hybridization

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

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

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

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

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

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