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Vibrationally Sensitive Nonlinear Optical Microscopy with Infrared Light UC Irvine UC Irvine Electronic Theses and Dissertations Title Vibrationally resonsant nonlinear optical microscopy with infrared light Permalink https://escholarship.org/uc/item/99v4r6tn Author Hanninen, Adam Publication Date 2018 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, IRVINE Vibrationally Sensitive Nonlinear Optical Microscopy with Infrared Light DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Physics and Astronomy by Adam M. Hanninen Dissertation Committee: Professor Eric O. Potma, Chair Professor Enrico Gratton Associate Professor Franklin Dollar 2018 Chapter 3 c 2017 Optical Society of America Chapter 4 c 2019 IEEE Chapter 5 c 2018 Optical Society of America All other materials c 2018 Adam M. Hanninen DEDICATION This thesis is proudly dedicated with heartfelt fondness to the family and friends who made my UCI grad school experience an absolute joy. Special gratitude goes to my mom and grandma, Linda and Verllien, and Bill, for supporting me to the core and always happy to talk, you are the best. I'm appreciative of my many friends: Alexey, Adam, Ethan, Hannah, Alex S., David, Trenton, Alex F., Alex H., Derek, Kyle, et alia for making my time here enjoyable and rememberable. To my friends in the Potma labs: Alba, Brian, Faezeh, Alex F., Bongsu, Ryan, John K., John H., Evan, Alex H., and of course the Prince- Richard, thank you for being excellent friends and reliable lab mates. An especially warm and gracious dedication to Olivia Humphrey, for going above and beyond in so many ways, you are truly special. Finally, to my advisor Eric, thank you for your patience, guidance, expertise, and sincere dedication to your students. You are a role model for us all. ii TABLE OF CONTENTS Page LIST OF FIGURES v LIST OF TABLES viii ACKNOWLEDGMENTS ix CURRICULUM VITAE x ABSTRACT OF THE DISSERTATION xii 1 Introduction to nonlinear optics 1 1.1 Development of nonlinear imaging in the mid-infrared . .1 1.2 Complimentary imaging techniques . .5 1.3 Theory of nonlinear light matter interaction . .8 2 Components of a SFG Microscope 17 2.1 Light source in the mid-infrared . 17 2.2 Optical components of the microscope . 20 2.3 Detection and signal processing . 25 2.3.1 Single photon counting circuit . 26 2.4 Superachromatic relay lenses . 30 2.4.1 Design of achromats . 31 3 Hyperspectyral Sum Frequency Generation Microscopy 40 3.1 Introduction . 40 3.2 Methods . 43 3.2.1 Light source . 43 3.2.2 Microscope . 44 3.2.3 Single photon counting . 45 3.2.4 Sample . 46 3.2.5 Image analysis . 47 3.3 Results . 47 3.3.1 Hyperspectral SFG reveals molecular orientation in fibrous structures 47 3.3.2 Hyperspectral SFG imaging of crystalline structures . 52 3.3.3 Multimodal SFG imaging . 53 iii 3.4 Discussion . 55 3.5 Conclusion . 58 4 Triple Modal Coherent Nonlinear Imaging With Vibrational Contrast 60 4.1 Coherent vibrational microscopy . 63 4.1.1 Probing vibrational coherences . 63 4.1.2 Selection rules . 64 4.1.3 Nonlinear susceptibilities . 65 4.1.4 Phase matching . 67 4.2 Nonlinear optical microscope for multi-modal vibrational imaging . 70 4.2.1 Light source . 70 4.2.2 Microscope . 72 4.2.3 Signal detection . 73 4.3 Imaging properties . 74 4.3.1 Spatial resolution . 74 4.3.2 Spectral dependence . 75 4.3.3 Image features . 80 4.4 IR-induced effects on CARS . 81 4.5 Triple-modal vibrational imaging . 83 4.6 Prospects and challenges . 83 4.7 Conclusion . 87 5 High Resolution Infrared Imaging of Biological Sampels with TSFG 88 5.1 Introduction . 88 5.2 Methods . 92 5.2.1 Light source . 92 5.2.2 Microscope . 92 5.2.3 Sample preparation . 94 5.3 Results . 94 5.3.1 Observation and characteristics of the TSFG signal . 94 5.3.2 Image features of TSFG microscopy . 97 5.3.3 TSFG microscopy of biological tissue samples . 98 5.4 Discussion . 99 5.5 Conclusion . 103 6 Future directions 106 6.1 Introduction . 106 6.2 Advancements in SFG . 107 6.3 Advancements in TSFG . 108 Bibliography 111 iv LIST OF FIGURES Page 1.1 Angled excitation geometry, SFG signal detected in reflection. This imple- mentation of a SFG microscope benefits from better phase matching, however it is not compatible with high NA objectives. .5 1.2 Collinear excitation inside the objective lens of a traditional inverted microscope.6 2.1 Diagram of single photon detection. When a signal exceeds a certain threshold a "count" value is given. In this case, orange shows a threshold too low which will have many counts, most of which are noise. Purple shows a threshold set too high, which misses most of the signal. Yellow is the ideal balance between detecting signals and rejecting noise. 27 2.2 Ray diagram and spot diagram of (top) CaF2 singlet lens and (bottom) CaF2 and sapphire doublet. The spot diagram shows the energy distribution of two wavelengths at focus. The singlet has poor performance while the doublet is entirely within the Airy disk which defines diffraction limited performance. 32 2.3 Shift in focal length as a function of wavelength for a f= 150mm plano-convex CaF2 lens (black) and a CaF2 and sapphire doublet (red). The doublet shows significant improved spectral performance by minimizing color dependent focal lengths. 33 2.4 x-axis in [ µm ] Transmission range of commercially available substrates in the MIR. Blue box indicates spectral range of interest. For a substrate to be considered its transmission must span over the entire blue box. 35 2.5 SFG signal from a GaP crystal using a CaF2 plano-convex singlet (black), and an air-spaced achromat (red). Signal with the doublet is much larger due to the tighter overlap in beam focus. Both lenses have approximately 180mm focal length. 37 2.6 Comparison of barium titanate images acquired in the SFG microscope using A. CaF2 singlets and B. air-spaced achromats for the scan and tube lenses. We see more signal and better contrast with the achromats. Field of view width and height- 80µm.............................. 38 2.7 Test plate images acquired in transmission using Left. CaF2 singlets NIR (top) MIR (bottom) and right. air-spaced doublets NIR (top) and MIR (bot- tom). With the singlets the two colors are out of focus due to chromatic shift, resulting in a blur. This aberration is corrected for with the achromat, bringing both colors to a common focal distance. 39 v 3.1 Schematic of the SFG microscope. Yb+ oscillator seeds the OPO. Galvano- metric mirrors are part of an Olympus Fluoview 300 laser scanner, and exci- tation and collection optics are part of an Olympus IX71 frame. PMT: pho- tomultiplier, BF: bandpass filter, Obj: microscope objective, DM: dichroic mirror. 44 3.2 SHG and SFG imaging of rat tail tendon, a collagen I rich tissue. a) SHG image obtained with the polarization orientation of the incident beam parallel to the long axis of the collagen fibers. b) SHG image similar to a), but with the polarization orientation rotated by 90o. c) SHG polarization plot taken in the red region of interest shown in a). Direction refers to the polarization orientation of the incident beam. d) SFG image at 2945 cm−1 of the same sample and with the polarization orientation of both beams aligned with the main axis of the collagen tissue. e) SFG image similar to e), but with the polarization orientation rotated by 90o. f) SFG spectra extracted from the hyperspectral data stack. Red spectrum refers to the region of interest in d) and blue spectrum refers to the region of interest indicated in e). Scale bar is 15 µm........................................ 49 3.3 Multivariate analysis of collagen rich tissue. a) Vertex component analysis (VCA) image showing three end-members in red, green and blue, based on a hyperspectral SFG data stack. The arrow indicates the polarization direction of the incident beams. b) Corresponding end-member SFG spectra extracted from the VCA. 50 3.4 Hyperspectral SFG imaging of a cellulose fiber. a) SFG image taken at 2945 cm−1. b) Corresponding SHG image. c) VCA image of the SFG hyperspectral data stack, showing three end-members in red, green and blue. d) SFG end- member spectra obtained from the VCA. Scale bar is 10 µm. 51 3.5 SFG imaging of cholesterol microcrystals. a) SFG image at 2845 cm−1. Scale bar is 10 µm. b) SHG polar plots obtained in the red and blue boxed regions of interest of the image in a). c) SFG spectra extracted from the regions of interest in image a). The red spectrum is obtained from the red box, whereas the blue spectrum is obtained from the blue box. d) Composite SFG image formed by overlaying images taken at 2955 cm−1 (blue), 2925 cm−1 (green), and 2845 cm−1 (red). 53 3.6 Cholesterol microcrystals visualized with a) SFG and b) CARS. Vibrational driving frequency in both images is set at 2845 cm−1. Scale bar is 10 µm. 55 4.1 Jablonski diagrams of the (a) SFG, (b) CARS and (c) TSFG processes. 64 4.2 Schematic of the triple-modal vibrational microscope system. The synchronously- pumped OPO delivers three beams (!p, !S and !IR) that are properly con- ditioned, delayed and collinearly combined on dichroic mirrors. The imaging platform is a modified laser-scanning microscope, optimized for NIR and MIR throughput. The condenser lens and detection bandpass filters are not explic- itly shown in this schematic.
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