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 singlecell 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
2D images
SHG
3D stack image
TPF
OVERLAY
Setup
Dr. Yeh’s Research Group Laser
TPF detector
SHG detector
Galvanometer driven mirror
Ultrafast laser
Objective
mh
b
Y
nt
Intensity (a.u.)
Y
Y
mh
b
350
500
650
Wavelength (nm)
Two photon microscopy
• Two photons both
excite and detect
specific gene patterns
Y
350
500
650
Wavelength (nm)
1 photon
One-Photon Excited
Fluorescence (OPEF)
Two-Photon Excited
Fluorescence (TPEF)
v.
2 photon
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: transgenes can be difficult to
induce
Linear Unmixing
Linear unmixing
Measurement is a linear sum of
constituent spectra
Ax
+Bx
AF ref
+Cx
eGFP
ref
= fit
citrine ref
measurement
sse=Σ(measurement-fit)2
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