\documentclass[style=fyma,paper=screen,mode=present,size=10pt]{powerdot} \usepackage{graphicx} \usepackage{amsmath} \usepackage{amssymb} \usepackage{braket} \DeclareGraphicsExtensions{.pdf, .jpg} \pdsetup{lf=Oral examination} \title{Research in \textit{in vivo}, trans-membrane potential measurement with second-harmonic generation techniques} \author{Nick Chernyy} \date{April 13th, 2007} \begin{document} \maketitle \begin{slide}{Outline} % \footnotesize % \tableofcontents \begin{itemize} \item Bio-potentials \item Optical techniques \item Second harmonic generation \item Proposed research \item Reference \end{itemize} \end{slide} \section{Bio-potentials} \begin{slide}{Background} What are bio-potentials? \begin{itemize} \item Cells maintain ionic gradients \item Ions are also used for communication and computation \item Ionic gradients lead to electric (Nernst) potentials \end{itemize} So why are they important? \begin{itemize} \item Changes in electric potential can be related to activity \item Potential distribution can be used to determine cell function \item Functional imaging can lead to a better understanding of biological systems \end{itemize} \end{slide} \begin{slide}{``Point'' electrodes} \twocolumn[lcolwidth=2.25in, rcolwidth=1in]{ Benefits: \begin{itemize} \item Easy to create and position \item Minimal instrumentation to operate \item Well-developed electro-chemistry for some electrodes \end{itemize} Deficiencies: \begin{itemize} \item Require intimate contact with tissue \item Only provide point measurements \item Miss the ``\textit{big picture}'' \end{itemize} }{ \begin{figure} \includegraphics[bb=0.0 0.0 313.0 308.0,width=1in]{./img/pipette.pdf} \end{figure} \begin{figure} \includegraphics[bb=0.0 0.0 701.0 501.0,width=1in]{./img/utah-array.pdf} \end{figure} \tiny{University of Utah} } \end{slide} \section{Optical techniques} \begin{slide}{Wide-field fluorescence imaging} \twocolumn[lcolwidth=2.55in, rcolwidth=1in]{ Separate excitation (shorter) and emission (longer) wavelengths. \linebreak[3] \begin{itemize} \item Wide range of dyes/markers available \item Emission and excitation can be split \item Possible to image structure as well as function \item Shallow penetration depth \item Direct/indirect tissue damage \item Diffraction limited \item Radiated photons incoherent \end{itemize} }{ \begin{figure} \includegraphics[bb=0.0 0.0 225.75 206.25,width=1.15in]{./img/fluor.pdf} \end{figure} \begin{figure} \includegraphics[bb=0.0 0.0 242.25 264.75,width=1.15in]{./img/f-diag.pdf} \end{figure} \tiny{Laboratory for Neuroengineering, Georgia-Tech.} } \end{slide} \begin{slide}{Confocal imaging} \small \twocolumn[lcolwidth=2.25in, rcolwidth=1in]{ \begin{itemize} \item ``Point'' excitation and sensing \item Improved spatial resolution \item Reduced depth of field \item Requires scanning \item Energy is localized \item Emission still incoherent \end{itemize} }{ \begin{figure} \includegraphics[bb=0.0 0.0 488.64 792.0,width=1.15in]{./img/confocal.pdf} \end{figure} \tiny{Structural image, Wadsworth Center} } \end{slide} \begin{slide}{Multi-photon confocal} \twocolumn[lcolwidth=1.5in]{ \small \begin{itemize} \item Multiple, lower-energy photons absorbed at the same time\footnote{``Same time'' is considered to be within a 0.1 femtosecond window.} \item Deeper penetration \item Improved resolution \end{itemize} }{ \begin{figure} \includegraphics[bb=0.0 0.0 361.0 414.0,width=2.2in]{./img/multi.pdf} \end{figure} \tiny{Nikon Corporation} } \end{slide} \section{Second harmonic generation (SHG)} \begin{slide}{Basic theory} \small Local, static electric field can modulate second-harmonic generation in response to incident radiation. Emission is coherent. \begin{center} \begin{equation} \label{eq:efield} \widetilde{E}(t)=Ee^{-i\omega t}+E^{*}e^{i\omega t} \end{equation} \begin{equation} \label{eq:expan} \widetilde{P}(t)= \chi^{(1)}\widetilde{E}(t)+ \chi^{(2)}\widetilde{E}^2(t)+ \chi^{(3)}\widetilde{E}^3(t)+ \cdots \end{equation} \begin{equation} \label{eq:expan2} \widetilde{P}^{(2)}(t)=\chi^{(2)}\widetilde{E}^2(t) =\chi^{(2)}(\underbrace{2EE^{*}}_{\textrm{static field}}+\underbrace{Ee^{-2i\omega t}+\textrm{c.c.}}_{\textrm{second harmonic}}) \end{equation} \begin{equation} \label{eq:wave} \nabla^2\widetilde{E}-\frac{n^2}{c^2} \frac{\partial^2\widetilde{E}}{\partial t^2} =\frac{4\pi}{c^2} \frac{\partial^2\widetilde{P}}{\partial t^2} \end{equation} \end{center} \end{slide} \begin{slide}{Susceptibility tensor} For a non-zero second harmonic generation, the second-order susceptibility tensor $\chi^{(2)}_{ijk}$ must be non-centrosymmetric. There must be anisotropy. \newline \newline Result of centrosymmetry: \begin{equation} \label{eq:centro1} \widetilde{E}^2(t) \to +\widetilde{E}^2(t): \widetilde{P}^{(2)}(t)=\chi^{(2)}\widetilde{E}^2(t) \end{equation} \begin{equation} \label{eq:centro2} \widetilde{E}^2(t) \to -\widetilde{E}^2(t): -\widetilde{P}^{(2)}(t)=\chi^{(2)}(-\widetilde{E})^2(t) \chi^{(2)}\widetilde{E}^2(t) \end{equation} \begin{equation} \label{eq:centro3} -\widetilde{P}^{(2)}(t)=\widetilde{P}^{(2)}(t) \end{equation} \begin{equation} \label{eq:centro4} \widetilde{P}^{(2)}(t)=0 \therefore \chi^{(2)}=0 \end{equation} \end{slide} \begin{slide}{Source of anisotropy} The most common sources of non-centrosymmetry in organic molecules are $\pi$ bonds \footnote{$\pi$ bonds are covalent bonds where lobes of adjacent electron orbitals overlap.} (G\"unter 2000).\linebreak[3] \begin{itemize} \item Bond electron donor (D) with acceptor (A) \item Higher energy to move electron from acceptor to donor \item Lack of symmetry results in anisotropy \end{itemize} \twocolumn[lcolwidth=1in,rcolwidth=4in]{ \begin{figure} \includegraphics[bb=0.0 0.0 307.0 139.0,width=1.25in]{./img/pibond.pdf} \end{figure} }{ $\begin{array}{ccc} \vec E \to & \vec E = 0 & \vec E \gets \\ D^{-} \leftrightsquigarrow A^{+} & D^{0} \leftrightsquigarrow A^{0} & D^{+} \leftrightsquigarrow A^{-} \\ \textrm{state } \ket{2} & \textrm{state } \ket{0} & \textrm{state } \ket{1} \end{array}$ } \end{slide} \begin{slide}{Available dyes} Molecules are typically non-polar (hydrophobic) therefore they enter lipid bi-layers readily, but also require non-polar solvents.\linebreak[3] \begin{tabular}[]{|c|c|c|} \hline molecule & solvent & $\lambda$ \footnote{Excitation wavelength (nm)} \\ \hline di-4-ANEPPS & Ethanol* & 850 \\ \hline RETINAL & Dimethyl Sulfoxide* & 850 \\ \hline MNA & Dioxane* & 1,064 \\ \hline DNAS & Chloroform* & 1,907 \\ \hline ANDS & Acetone* & 1,064 \\ \hline \end{tabular}\linebreak[3] *Will also dissolve cell membranes! \end{slide} \section{Proposed research} \begin{slide}{Need for better dyes} \normalsize{Requirement for organic solvents makes staining live tissue difficult.}\linebreak[3] \begin{figure} \includegraphics[bb=0.0 0.0 530.0 530.0,width=2.25in]{./img/sh-cells.pdf} \end{figure} \tiny{Structural imaging of lymphatic mouse cells. Fibers are on the order of 1$\mu$m. Harvard.} \end{slide} \begin{slide}{Or better placement methods} Injecting SHG dyes into cellular membranes without damage is difficult, what if we get someone else to do it?\linebreak[4] A successful candidate must: \begin{itemize} \item Have experience assembling organic molecules \item Be able to follow directions \item Be ready to work for peanuts \end{itemize} \end{slide} \begin{slide}{Perfect candidate} Why not use the cells themselves?\linebreak[3] The cells: \begin{itemize} \item Have been manipulating proteins since they were created \item Better than PPM mutation rates, and then safety checks in protein sequencing \item Require carbohydrates, proteins and fats for operation (Peanuts) \end{itemize} The hard part is getting the cells to make custom proteins. \end{slide} \begin{slide}{Computational search} \twocolumn[lcolwidth=2.5in,rcolwidth=1in]{ Steps:\small \begin{itemize} \item Identify target cells \item Retrieve protein conformation information \item Divide into sub-domains \item Compute second order susceptibility \item Locate natural proteins with good SHG properties \item Look for amino-acid replacements to enhance SHG \item Generate new DNA sequence \end{itemize} End goal is to modify target's genome with new DNA sequence }{ \begin{figure} \includegraphics[bb=0.0 0.0 376.5 516.75,width=1in]{./img/protein.pdf} \end{figure} } \end{slide} \begin{slide}{Final deliverables} \twocolumn[lcolwidth=2.5in,rcolwidth=1.1in]{ \begin{itemize} \item Animals exhibit SGH-protein expression \item Animals that are robust enough to be used in research \item Better signal resolution than fluorescence \end{itemize} }{ \begin{figure} \includegraphics[bb=0.0 0.0 399.0 450.0,width=1.1in]{./img/fl-fish.pdf} \end{figure} \begin{figure} \includegraphics[bb=0.0 0.0 100.0 75.38,width=1.1in]{./img/fl-mouse.pdf} \end{figure} } \end{slide} \begin{slide}{Hard work ahead} \begin{itemize} \item Computation of energy states required \item Comparable to fluorescence modeling \item Limited amino-acid substitutions without ill effects \item Genetic manipulation is extremely difficult (GFP animals from previous slide took decades to develop) \end{itemize} \end{slide} \begin{slide}{Required resources} \small This research is inter-disciplinary in nature, so collaborations are a must. \begin{itemize} \item Computers with software \begin{itemize} \item Modification of Folding@Home distributed software possible \item Computers will have to be purchased for simulation \item Possible to also use spare CPU cycles like Folding@Home \end{itemize} \item Microscope capable of SHG measurement \begin{itemize} \item Confocal microscope required (Available at ESM and Huck Institute) \item Fast laser also required, possibly available at ESM \end{itemize} \item Biological expertise \begin{itemize} \item Cooperation with researchers from Life Sciences will be required for genetics work \end{itemize} \end{itemize} \end{slide} \section{References} \begin{slide}{References - Thank you for listening!} \footnotesize \begin{thebibliography}{1} \bibitem{ref:Bouevitch} Bouevitch. O \textit{et al.}, Probing membrane potential with non-linear optics, Biophysical J. 65: 1993 p. 672-679 \bibitem{ref:Boyd} Boyd, R. W., Nonlinear optics, Academic Press, San Diego, CA 1992 \bibitem{ref:Gunter} G\"unter, P., Nonlinear optical effects and materials, Springer Press, Berlin, Germany 2000 \bibitem{ref:Herskind} Herskind, P \textit{et al.}, Second harmonic generation of light at 544 and 272 nm from an ytterbium-doped distributed fiber laser, Optics Letters 32 (3): 2007 p. 268-270 \bibitem{ref:Malmivuo} Malmivuo, J., Plonsey, R., Bioelectromagnetism - principles and applications of bioelectric and biomagnetic fields, Oxford University Press, New York, NY 1995 \bibitem{ref:Millard} Millard, A. C. \textit{et al.}, Direct measurement of the voltage sensitivity of second harmonic generation from a membrane dye in patch-clamped cells, Optics Letters 28 (14): 2003 p. 1221-1223 \bibitem{ref:Mutso} Mutso, N. \textit{et al.}, Imaging membrane potential in dendtritic spines, PNAS 13 (3): 2006 p. 786-790 \bibitem{ref:Nemet} Nemet, B. A., Nikolenko, V., Yuste, R., Second harmonic imaging of membrane potential of neurons with retinal, J. Biomed. Optics 9 (5): 2004 p. 873-881 % \bibitem{ref:Pena} Pena, A. M. \textit{et al.}, Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multi-photon microscopy, Microscopy Res. and Technique 70: 2007 p. 162-170 % \bibitem{ref:Schenke} Schenke-Layland, K. \textit{et al.}, Two-photon microscopes and in vivo multiphoton tomographs - powerful diagnostic tools for tissue engineering and drug delivery, Advanced Drug Delivery Rev. 58: 2006 p. 878-896 \bibitem{ref:Vanzi} Vanzi, F. \textit{et al.}, New techniques in linear and non-linear laser optics in muscle research, J. Muscle Res. Cell Motil. 27: 2006 p. 469-479 \end{thebibliography} \end{slide} \end{document}