Duke University, Fitzpatrick Institute for Photonics BIOS Lab, Department of Biomedical Engineering Advances in time-resolved and spectroscopic quantitative phase microscopy Duke University Department of Biomedical Engineering Matthew Rinehart Preliminary Examination 7/24/2012

Outline Summary of course work Peer-reviewed publications Motivation & introduction Preliminary results –QPM technology –QPM applications –QPS Research aims & timeline

Summary of course work TermCourseCourse TitleInstructorUnits Fall 2008PATH 225 Intro to Systemic HistologyL. Hale2 BME 265Advanced OpticsJ. Kim3 BME 233Modern Diagnostic Imaging Systems J. MacFall3 Spring 2009 ECE 299Imaging & SpectroscopyD. Brady3 PATH 250 General PathologyL. Hale4 Fall 2009ECE 299Holography & Coherent ImagingD. Brady3 PHY 230Math Methods in PhysicsA. Kotwal3 Spring 2010 BME 265Image ProcessingS. Farsiu3 BIO 154Fundamentals of NeuroscienceS. Bilbo3 Fall 2010ECE 376Lens DesignD. Brady3 Spring 2011 ECE 399Independent Study: Broadband Diffraction Phase Microtomography D. Brady3 Fall 2011BME 234Modern MicroscopyA. Wax3 BME 362Invention to ApplicationB. Myers3 Spring 2012 BME 362Invention to ApplicationB. Myers3 RequirementsUnits Advanced Math3 Life Sciences8 ECE Masters Courses 12 Total:42 RCR Hours8 (of 12) TermCourseCourse TitleInstructor Fall 2009BME 171Intro to Signals & SystemsJ. Izatt Fall 2010BME 171Intro to Signals & SystemsJ. Izatt Teaching Experience

Publications Peer-reviewed journal articles 1.S. Kim, M. T. Rinehart, H. Park, Y. Zhu, and A. Wax, “Spectrally multiplexed photothermal OCT and novel detection methods," Biomedical Optics Express (2012). In Review. 2.A. Wax, M. Giacomelli, T. E. Matthews, M. T. Rinehart, F. E. Robles, Y. Zhu, “Optical Spectroscopy of Biological Cells,” Advances in Optics and Photonics, In Press. 3.M. T. Rinehart, Y. Zhu, and A. Wax, “Quantitative phase spectroscopy," Biomedical Optics Express 3, 958 (2012). 4.M. T. Rinehart, T. K. Drake, F. E. Robles, L. C. Rohan, D. Katz, and A. Wax, “Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy," Journal of Biomedical Optics 16, (2011). 5.N. G. Terry, Y. Zhu, M. T. Rinehart, W. J. Brown, S. C. Gebhart, S. Bright, E. Carretta, C. G. Ziefle, M. Panjehpour, J. Galanko, R. D. Madanick, E. S. Dellon, D. Trembath, A. Bennett, J. R. Goldblum, B. F. Overholt, J. T. Woosley, N. J. Shaheen, A. Wax, “Detection of Dysplasia in Barrett's Esophagus With In Vivo Depth- Resolved Nuclear Morphology Measurements,” Gastroenterology – 20 (2010) 6.M. T. Rinehart, N. T. Shaked, N. J. Jenness, R. L. Clark, and A. Wax, "Simultaneous two-wavelength transmission quantitative phase microscopy with a color camera," Opt. Lett. 35, (2010) 7.J. C. Booth, N. D. Orloff, J. Mateu, M. D. Janezic, M. T. Rinehart, and J. A. Beall, “Quantitative Permittivity Measurements of Nanoliter Liquid Volumes in Microfluidic Channels to 40 GHz,” IEEE Transactions on Instrumentation and Measurement 99, 1-10 (2010) 8.N. T. Shaked, Y. Zhu, M. T. Rinehart, and A. Wax, “Two-step-only phase-shifting interferometry with optimized detector bandwidth for microscopy of live cells,” Opt. Express 17, (2009) 9.N. T. Shaked, M. T. Rinehart, and A. Wax, "Dual-interference-channel quantitative-phase microscopy of live cell dynamics," Opt. Lett. 34, (2009) Book Chapters 1.Yizheng Zhu, Matthew T. Rinehart, Francisco E. Robles, and Adam Wax, “Polarization and Spectral Interferometric Techniques for Quantitative Phase Microscopy,” in Biomedical Optical Phase Microscopy and Nanoscopy, Shaked (Editor), Elsevier, In press. 2.N. T. Shaked L. L. Satterwhite, M. T. Rinehart, and A. Wax, "Quantitative Analysis of Biological Cells Using Digital Holographic Microscopy," in Holography, Research and Technologies, Rosen (Editor), Intech, N. T. Shaked, M. T. Rinehart, and A. Wax, "Quantitative phase microscopy of biological cell dynamics by wide-field digital interferometry," in Coherent Light Microscopy: Imaging and Quantitative Phase Analysis, Ferraro, Wax and Zalevsky (Eds.), Springer, 2011.

Publications +14 conference abstracts/presentations (incl. SPIE) +2 additional non-reviewed pubs

Motivation Semitransparent objects are hard to image While methods exist to produce high-contrast images of some of these objects, these methods are not well-suited for quantitative analysis Advanced holographic imaging techniques have been developed for quantitative imaging of this class of samples, but have not yet become useful to biology/clinical researchers Motivation: Develop techniques and instruments aimed at making QPM more accessible to laboratories as a research tool for investigating biological samples

Interferometrically measuring optical phase delays can yield quantitative information Optimal for nearly transparent samples that lack inherent contrast Resulting data can be both visualized in quasi-3D and also used for quantitative analysis Quantitative phase microscopy Phase microscopy measures relative optical path delays, proportional to refractive index mismatch ΔϕΔϕ

QPM for biological samples QPM has been developed to study quantitative changes in in vitro cell cultures Morphological parameters used by cell biologists are based on cell thickness rather than phase profile –E.g. – cell volume, cell force distribution How to decouple refractive index from thickness using phase profiles? These methods have limited use for dynamically changing cells with organelles with non-uniform refractive index 1.Assume homogeneous refractive index 2.Make differential measurements using multiple wavelengths or varying-RI media 1.Assume homogeneous refractive index 2.Make differential measurements using multiple wavelengths or varying-RI media 3.Inhomogeneous refractive index (organelles) Can still extract parameters Relative area, dry mass, volume, average phase Use multiple wavelengths, use multiple media with different RIs

QPM for biological samples QPM has been developed to quantitatively investigate morphological structures and dynamic changes in in vitro cell cultures –Cell volume –Cell force distribution –Organelle location Previous work has relied on quantitative analysis of temporal and spatial fluctuations Relatively little attention has been focused on quantitative spectral measurements –Material dispersion properties –Absorption effects via complex refractive index 1.Assume homogeneous refractive index 2.Make differential measurements using multiple wavelengths or varying-RI media 3.Inhomogeneous refractive index (organelles) Can still extract parameters Relative area, dry mass, volume, average phase Use multiple wavelengths, use multiple media with different RIs 9

Specific Aims 1)Optimize QPM for analysis of biological systems 2)Apply QPM to biologically-relevant systems 3)Extend QPM to quantitative phase spectroscopy (QPS) 4)Advanced implementations and applications of QPM & QPS

Specific Aims Develop QPM instrumentation & techniques Develop QPS instrumentation & techniques Apply QPM to dynamic systems Develop QPS for molecular specificity

Specific Aims Develop QPM instrumentation & techniques Develop QPS instrumentation & techniques Apply QPM to dynamic systems Apply QPS for molecular specificity

QPM Instrumentation Microscope configuration Microscopic samples delay light 4F configuration images wavefront onto camera Samples are relatively thin

QPM Instrumentation reference field & interference 14 matched wavefronts via matched objectives

On-axis phase shifting vs. off-axis Phase-shifting interferometry: can require mechanical shifting Dynamic Interferometry, Neal Brock, James C. Wyant, et al. Proceedings of SPIE Vol (SPIE, Bellingham, WA), page 58750F-1, 2005

QPM: Off-axis Theory Currently examining phase; could recover amplitude too { } =atan2

Phase, RI, thickness, wavelength

QPM spatial frequency comparisons Natan T. Shaked, Yizheng Zhu, Matthew T. Rinehart, and Adam Wax Optics Express, Vol. 17, Issue 18, pp (2009)

Result 1: Dynamic SOFFI N.T. Shaked, M.T. Rinehart, and A. Wax, "Dual-interference-channel quantitative-phase microscopy of live cell dynamics," Opt. Lett. 34, (2009)

Beating rat cardiomyocyte MDA-MB-468 human breast cancer cell Acquisition rate: 120 frames per second N.T. Shaked, M.T. Rinehart, and A. Wax, "Dual-interference-channel quantitative-phase microscopy of live cell dynamics," Opt. Lett. 34, (2009) Experimental Results High-speed videos

Phase Unwrapping

2π Rollover, 2λ Unwrapping 2 π rollover can be removed using unwrapping algorithms –Iterative gradient-minimization algorithms Sharp changes in phase cannot be accurately resolved Imaging at 2 wavelengths extends measurement range 633nm & 532nm  3.33 μ m m = ? Subtract wrapped phase maps & add 2π where <0 Amplifies phase noise Use beat wavelength phase map as guide to add correct multiple of 2 π

2λ Phase Unwrapping Result

Phase Referencing

film Phase unwrapping & RI mapping Inhomogeneity of films can cause spatial variation in phase much greater than π Use of temporal + spatial phase unwrapping together to ensure –Assumes that the phase does not change by more than π between time points Phase Reference for RI mapping Need a tool for full decoupling of refractive index from height in thick samples We have designed a PDMS ramp to provide an absolute refractive index The ramp is positioned on the edge of the field of view FOV

Calculating RI from phase μm

Conclusions & next steps Develop QPM instrumentation & techniques Conclusions Examined tradeoff between on-axis & off- axis in phase microscopy Developed balanced approach with simultaneous acquisition Developed single-shot 2-wavelength QPM instrumentation & method with color camera Came up with system for calibrating a chamber for absolute imaging refractometry Next Steps Build turnkey device that is robust and user-friendly Standardized sample loading Basic software that enables simple image processing

Specific Aims Develop QPM instrumentation & techniques Develop QPS instrumentation & techniques Apply QPM to dynamic systems Apply QPS for molecular specificity

© 2006 All Rights Reserved, Conrex Pharmaceuticals Corp. Microbicidal films Parameters of interest swelling index bioadhesion properties moisture content disintegration time dissolution & drug release drug content uniformity Current method of evaluation Franz cells are used to measure membrane / tissue permeability. Bulk parameters are calculated, but the model assumes homogeneity Quantitative phase microscopy can be used to study microbicidal film dissolution dynamics with high temporal and spatial resolution Films as a drug delivery vehicle portable easy to store discreet low cost require no applicator stored in solid form, can chemically stabilize drugs over long periods of time (AP Photo/Keith Srakocic)

Preliminary Dissolution Results Bolus injection of water in gel

Film imaging geometry Microbicidal films characteristics: Loaded with active agent CSIC μm thick 4-10 minute disintegration time Refractive index = 1.44 Films are highly inhomogeneous Common ingredients: of EDTA, PVA, Carbopol 974, cholesterol, ethanol, BHA, glycerin, active agent Experimental Parameters 10x magnification ~500x700um FOV Flood chamber with water “instantaneously” Acquire images every 0.5-1s as film dissolves

Microbicidal film refractive index Refractive Index RI % change 50 % -50 % 0 % Assumes one area in FOV is always a known RI (here, water)

Calibrated film refractometry Calibration ramp structure allows absolute RI calculation of the chamber Combination spatial + temporal phase unwrapping allows film RI to be determined even during initial hydration chaotic changes

Quantitative analysis of dissolution

Experiment considerations Only a small area of the film is within the field of view The film area is flooded from the side at t=0 –Doesn’t necessarily match biophysical hydration of films Model assumes homogeneous film RI Data is 2D RI map; 3D mapping would be ideal

Conclusions & next steps Conclusion QPM Refractometry of films offers a quantitative tool for measuring dissolution characteristics RI % hydration, water Total disintegration time Next Steps Develop quantitative summary metrics & standardized method for comparing dissolution across different films Thickness Composition Develop methods of measuring active agent / particle / molecule release from films Fluorescence Absorptivitiy Raman Apply QPM to dynamic systems

Specific Aims Develop QPM instrumentation & techniques Develop QPS instrumentation & techniques Apply QPM to dynamic systems Apply QPS for molecular specificity

Why Spectroscopy? Digitally adjust coherence properties of sample illumination –Improve noise characteristics –Synthesize coherence windows to gain depth sensitivity Measure sample dispersion Molecular identification based on Kramers- Kronig relation (concentration-dependent RI phenomena)

Quantitative phase spectroscopy We have developed a novel quantitative phase microscope with a high power rapidly tunable narrowband source to perform high resolution quantitative phase spectroscopy. 39 Quantitative phase microscopy Rapidly-tunable laser source Quantitative phase spectroscopy

QPS outline Techological Advances –Illumination temporal sweep –Low coherence illumination Results –Coherent noise reduction –RI Spectroscopy of absorptive features –Refractometry for concentration measurement

Spectral Sweep of Fianium+AOTF 41

QPS: low-coherence illumination

Low-coherence illumination effects 43

QPS

Spectroscopic imaging results Speckle reduction via averaging Dispersion measurements of discrete fluorophore objects Bulk measurements of homogeneous fluid samples 45

Transmission phase target custom-molded with PDMS 90nm nominal thickness Average phase images acquired across the spectrum Coherent noise reduction mrad  11.8mrad σ(Δφ)σ(ΔOPL) 38.2 mrad3.55 nm11.8 mrad1.09 nm 10μm

Microsphere dispersion measurements Add: KK theory Speckle reduction comparison? 48 polystyrene + fluorophore Add kramers kronig graph here

Bulk hemoglobin RI measurements 49 DiH 2 O PDMS Δφ ΔnΔn PDMS 331 g/L 165 g/L 110 g/L 55 g/L H2OH2O Oxyhemoglobin (HbO 2 ) 10 μm σ =

Conclusions & next steps Develop QPS instrumentation & techniques Instrumentation Advances Illumination temporal sweep Low coherence illumination Techniques Demonstrated Coherent noise reduction RI Spectroscopy of absorptive features Refractometry for concentration measurement Next Steps Improve spectral illumination characteristics Add trinocular display

Specific Aims Develop QPM instrumentation & techniques Develop QPS instrumentation & techniques Apply QPM to dynamic systems Develop QPS for molecular specificity

Quantitative Phase Spectroscopy y.k. Park, spectroscopic analysis of blood

Proposed Work Develop QPM instrumentation & techniques Develop QPS instrumentation & techniques Apply QPM to dynamic systems Develop QPS for molecular specificity Build InCh Microscope Develop robust & standardized assay for film dissolution Improve Illumination Add binocular display Use ratiometric imaging & photothermal Image microbicidal films & RBCs with molecular specificity

Dual Window?? Would go under QPS techniques & Instrumentation Don’t really want to go into this much Would extend coherent averaging to be adjustable (digital coherence refocusing)

Proposed Work QPM Instrumentation Portable device High-throughput cell flow device QPM Application Film dissolution assay RBC/parasite assay QPS Instrumentation & methods Improved FOV & illumination User-friendly microscope –Binocular display –Software development DW method for digital coherence refocusing Molecular contrast –Photothermal –Ratiometric imaging

InCh microscope Current microscopy of blood smears search for morphology & parasites Requires staining & trained microscopist reading slides NOT high throughput Suffers from interobserver variability InCh microscope would be a robust platform for automated image acquisition & analysis NSF I-Corps program: assess commercial viability, connect the correct value proposition to the customer needs

Microbicidal films (AP Photo/Keith Srakocic) Develop quantitative summary metrics to create a standardized assay for evaluating film dissolution Evaluate various films w/ differring compositions, thicknesses, etc. Develop quantitative summary metrics & standardized method for comparing dissolution across different films Thickness Composition Conduct study of dissolution across multiple films to validate utility of method Only a small area of the film is within the field of view The film area is flooded from the side at t=0 Doesn’t necessarily match biophysical hydration of films Model assumes homogeneous film RI Data is 2D RI map; 3D mapping would be ideal

QPS Instrumentation Advances Narrower-band illuminationIncorporate Trinocular Display –User-friendly for sample alignment before/during/after data acquisition

QPS Molecular Specificity Lab has developed methods of photothermal excitation with phase detection (REF TO SANGHOON KIM PAPER); demonstrated with OCT. Should be usable with QPS w/o a problem Previously examined fluorescence microscopy + phase microscopy –Not too hot – low SNR on fluorescence, not optimized for high sensitivity, limited in time. Fluorophore signatures maybe? –Limited to fluorescence, can’t see intrinsic absorption Ratiometric imaging demonstrated by Fu, et al. –Exploits dispersion as contrast –Extend to use dispersion, endogenous absorption, and additive dyes –No photobleaching –high SNR possible Hb HbO 2 Measure differences in oxygenation of bulk hemoglobin / blood solutions Potentially measure oxygenation of individual cells? Molecular specificity and digital coherence refocusing in quantitative phase spectroscopy M. Rinehart & A.Wax Quantitative phase spectroscopy (QPS) has been developed as a method of investigating the wavelength-dependent refractive index of semitransparent microscopic samples with high spectral and spatial resolution. Here, we present novel wavelength illumination patterns for acquiring holographic data as well as algorithms for digital coherence refocusing and ratiometric imaging of multiple absorptive species in a single sample. Light from a supercontinuum source spanning from 450nm to 750nm is filtered with a rapidly-tunable custom spectral filter to a bandwidth of 100Hz) using a sparse set of scanned illumination wavelengths. We will present validation results of these novel techniques by imaging thick samples with embedded absorptive objects and also samples with multiple absorbers present. Targeted Delivery of PSC-RANTES for HIV-1 Prevention using Biodegradable Nanoparticles. Ham, et al. Pharmaceutical Research, Vol. 26, No. 3, March 2009

Combined FPM system Transmission quantitative phase Blue fluorescence excitation Red fluorescence excitation Common imaging pathway Photometrics CoolSNAP cf

System filters & fluorophores Bandpass filter Chroma Technology notch filter > OD5 at both 376nm and 632.8nm < 95% transmission nm, nm Fluorophore choice Nucleic acid stain (Invitrogen Hoescht 34580) Mitochondria stain (Invitrogen MitoTracker Deep Red)

Fluorescence imaging channel We have measured fluorescence of films and active agents (UC-781, CSIC) Fluorescence can potentially be used to simultaneously investigate the structure of these films or the rate of diffusion of active agents Targeted Delivery of PSC-RANTES for HIV-1 Prevention using Biodegradable Nanoparticles. Ham, et al. Pharmaceutical Research, Vol. 26, No. 3, March 2009

System filters & fluorophores Bandpass filter Chroma Technology notch filter > OD5 at both 376nm and 632.8nm < 95% transmission nm, nm Fluorophore choice Nucleic acid stain (Invitrogen Hoescht 34580) Mitochondria stain (Invitrogen MitoTracker Deep Red)

Coregistered fluorescence & quantitative phase 10μm Fluorescence Map Red = Mitochondria Blue = Nuclei Quantitative phase profile Phase Instantaneous Differential (PID) Blue= PID + Red = PID -

Proposed Work Develop QPM instrumentation & techniques Develop QPS instrumentation & techniques Apply QPM to dynamic systems Apply QPS for molecular specificity Build InCh Microscope Develop robust & standardized assay for film dissolution Improve Illumination Add binocular display Use ratiometric imaging & photothermal Image microbicidal films & RBCs with molecular specificity