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