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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. - PowerPoint PPT Presentation
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Non-linear Optical Microscopy:Viewing embryonic development of zebra fish
Esther JohnsonDel Rio High School – PhysicsDr. Alvin Yeh, Associate Professor of Biomedical EngineeringDr. 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 130. 9/7/2007. pp784-795
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 images 3D 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
350 500 650 350 500 650Wavelength (nm) Wavelength (nm)
Inte
nsi
ty (
a.u
.)
Y
Y Y
Y
nt
mhb
mhb
One-Photon ExcitedFluorescence (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 UnmixingLinear unmixingMeasurement 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 ProgramNational Science FoundationNuclear Power InstituteTexas Workforce CommissionHolly GibbsDr. Arne LekvenDr. Alvin YehKirsten Brink