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Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections
Portland State University PDXScholar Dissertations and Theses Dissertations and Theses Fall 12-1-2017 Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections Fredrick Michael DeArmond Portland State University Let us know how access to this document benefits ouy . Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Physics Commons Recommended Citation DeArmond, Fredrick Michael, "Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections" (2017). Dissertations and Theses. Paper 4036. 10.15760/etd.5920 This Dissertation is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. For more information, please contact [email protected]. Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections by Fredrick Michael DeArmond A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Applied Physics Dissertation Committee: Erik J. Sánchez, Chair Erik Bodegom Ralf Widenhorn Robert Strongin Portland State University 2017 ABSTRACT As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes increased interest in and demand for robust non-linear fluorescent markers, and characterization of the associated tool set. -
Second Harmonic Imaging Microscopy
170 Microsc Microanal 9(Suppl 2), 2003 DOI: 10.1017/S143192760344066X Copyright 2003 Microscopy Society of America Second Harmonic Imaging Microscopy Leslie M. Loew,* Andrew C. Millard,* Paul J. Campagnola,* William A. Mohler,* and Aaron Lewis‡ * Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030-1507 USA ‡ Division of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel Second Harmonic Generation (SHG) has been developed in our laboratories as a high- resolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1- and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison. -
Replace This with the Actual Title Using All Caps
THREE-PHOTON MICROSCOPY AT 1700 NM FOR IN VIVO IMAGING A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Nicholas Geoffrey Horton May 2015 © 2015 Nicholas Geoffrey Horton THREE-PHOTON MICROSCOPY AT 1,700 NM FOR IN VIVO IMAGING Nicholas Geoffrey Horton, Ph. D. Cornell University 2015 Multiphoton fluorescence microscopy (MPM) allows scientists to noninvasively observe structures deep within tissue. Two-photon microscopy (2PM) in the 750-1000 nm excitation region has been the standard MPM technique since its first demonstration in 1990. However, the maximum imaging depth for 2PM is limited by the signal-to-background ratio (SBR). In this dissertation, three-photon imaging at 1700 nm excitation wavelength (1700 nm 3PM) is presented. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the SBR limitations of 2PM, enabling biological investigations to take place at greater depth within tissue. In chapter 1, tissue imaging is discussed, paying special attention to the development of laser scanning fluorescence microscopy. In chapter 2, non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using 1700 nm 3PM is presented. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. In chapter 3, dispersion compensation of 1700 nm 3PM is discussed. Signal generation in 3PM is proportional to the inverse-squared of the pulse width. We show that the high normal dispersion of a silicon wafer can be conveniently used to compensate the anomalous dispersion of a 1,700 nm excitation three-photon microscope. -
Fv1000 Fluoview
Confocal Laser Scanning Biological Microscope FV1000 FLUOVIEW FLUOVIEW—Always Evolving FLUOVIEW–—From Olympus is Open FLUOVIEW—More Advanced than Ever The Olympus FLUOVIEW FV1000 confocal laser scanning microscope delivers efficient and reliable performance together with the high resolution required for multi-dimensional observation of cell and tissue morphology, and precise molecular localization. The FV1000 incorporates the industry’s first dedicated laser light stimulation scanner to achieve simultaneous targeted laser stimulation and imaging for real-time visualization of rapid cell responses. The FV1000 also measures diffusion coefficients of intracellular molecules, quantifying molecular kinetics. Quite simply, the FLUOVIEW FV1000 represents a new plateau, bringing “imaging to analysis.” Olympus continues to drive forward the development of FLUOVIEW microscopes, using input from researchers to meet their evolving demands and bringing “imaging to analysis.” Quality Performance with Innovative Design FV10i 1 Imaging to Analysis ing up New Worlds From Imaging to Analysis FV1000 Advanced Deeper Imaging with High Resolution FV1000MPE 2 Advanced FLUOVIEW Systems Enhance the Power of Your Research Superb Optical Systems Set the Standard for Accuracy and Sensitivity. Two types of detectors deliver enhanced accuracy and sensitivity, and are paired with a new objective with low chromatic aberration, to deliver even better precision for colocalization analysis. These optical advances boost the overall system capabilities and raise performance to a new level. Imaging, Stimulation and Measurement— Advanced Analytical Methods for Quantification. Now equipped to measure the diffusion coefficients of intracellular molecules, for quantification of the dynamic interactions of molecules inside live cell. FLUOVIEW opens up new worlds of measurement. Evolving Systems Meet the Demands of Your Application. -
Wide. Fast. Deep. Recent Advances in Multi-Photon Microscopy of in Vivo Neuronal Activity
TechSights Wide. Fast. Deep. Recent Advances in Multi- Photon Microscopy of in vivo Neuronal Activity https://doi.org/10.1523/JNEUROSCI.1527-18.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1527-18.2019 Received: 2 March 2019 Revised: 27 September 2019 Accepted: 27 September 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 the authors 1 Wide. Fast. Deep. Recent Advances in Multi-Photon Microscopy of in vivo Neuronal Activity. 2 Abbreviated title: Recent Advances of in vivo Multi-Photon Microscopy 3 Jérôme Lecoq1, Natalia Orlova1, Benjamin F. Grewe2,3,4 4 1 Allen Institute for Brain Science, Seattle, USA 5 2 Institute of Neuroinformatics, UZH and ETH Zurich, Switzerland 6 3 Dept. of Electrical Engineering and Information Technology, ETH Zurich, Switzerland 7 4 Faculty of Sciences, University of Zurich, Switzerland 8 9 Corresponding author: Jérôme Lecoq, [email protected] 10 Number of pages: 24 11 Number of figures: 6 12 Number of tables: 1 13 Number of words for: 14 ● abstract: 196 15 ● introduction: 474 16 ● main text: 5066 17 Conflict of interest statement: The authors declare no competing financial interests. 18 Acknowledgments: We thank Kevin Takasaki and Peter Saggau (Allen Institute for Brain Science) for providing helpful 19 comments on the manuscript; we thank Bénédicte Rossi for providing scientific illustrations. -
Two-Photon Excitation Fluorescence Microscopy
P1: FhN/ftt P2: FhN July 10, 2000 11:18 Annual Reviews AR106-15 Annu. Rev. Biomed. Eng. 2000. 02:399–429 Copyright c 2000 by Annual Reviews. All rights reserved TWO-PHOTON EXCITATION FLUORESCENCE MICROSCOPY PeterT.C.So1,ChenY.Dong1, Barry R. Masters2, and Keith M. Berland3 1Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; e-mail: [email protected] 2Department of Ophthalmology, University of Bern, Bern, Switzerland 3Department of Physics, Emory University, Atlanta, Georgia 30322 Key Words multiphoton, fluorescence spectroscopy, single molecule, functional imaging, tissue imaging ■ Abstract Two-photon fluorescence microscopy is one of the most important re- cent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast im- ages and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine. CONTENTS INTRODUCTION ................................................ 400 HISTORICAL REVIEW OF TWO-PHOTON MICROSCOPY TECHNOLOGY ...401 BASIC PRINCIPLES OF TWO-PHOTON MICROSCOPY ..................402 Physical Basis for Two-Photon Excitation ............................ -
SPARQ-Ed Risk Assessment Sheet : Immunofluorescence
SPARQ-ed Risk Assessment Sheet : Immunofluorescence Hazard Analyse / Evaluate Risk Overall Risk Category Description of Risk Source Current Controls Event Category Consequences Exposure Probability (see explanation on last page) Exposure PPE worn (gloves, closed Prob footwear, and safety Chemical exposure - VR R U O F C Exposure to Chemical Agents : Risk of Unusual : goggles provided). Use body contact, spills Unusual but eye and skin exposure to chemicals Immuno- AC Low Low Low Low Mod Subs of potentially harmful and splash and Other Contact Minor : General Possible: Less likely (such as fixation / permeabilisation fluorescence QP Low Low Low Low Low Mod Chemical chemicals/substances in inhalation (eg. 4% with Chemical or chemicals used would to occur with the buffer containing 4% staining not a UP Low Low Low Low Low Low fume cupboard/safety paraformaldehyde is Substance be irritants only. control measures but paraformaldehyde) throughout the common cabinet. MSDS available. irritant to eyes and not impossible. RP Low Low Low Low Low Low staining procedure. procedure. Well documented skin). C Low Low Low Low Low Low procedures. PI Low Low Low Low Low Low Personal precaution Exposure procedures to be Prob followed in case of VR R U O F C Sharps : Immunoflourescence is injury. PPE worn Sharps injury (cuts or Unusual : AC Low Low Low Low Mod Subs generally performed with sections on (wearing gloves, closed slicing of fingers) Immuno- Conceivable : Sharps slides or with cells on coverslips. shoes, lab coat and from broken Hitting Objects Minor : May require fluorescence injury is likely to QP Low Low Low Low Low Mod Immunofluorescence staining of cells Mechanical safety goggles are coverslips or pointed with Part of first aid for minor cuts. -
Confocal Microscopy
Confocal microscopy Chapter in Handbook of Comprehensive Biophysics ( in press 2011) Elsevier Brad Amos MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH UK e-mail [email protected] Gail McConnell University of Strathclyde , Centre for Biophotonics 161 Cathedral Street , Glasgow G4 0RE UK [email protected] Tony Wilson Dept. of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK. eMail: [email protected] 1 Introduction A confocal microscope is one in which the illumination is confined to a small volume in the specimen, the detection is confined to the same volume and the image is built up by scanning this volume over the specimen, either by moving the beam of light over the specimen or by displacing the specimen relative to a stationary beam. The chief advantage of this type of microscope is that it gives a greatly enhanced discrimination of depth relative to conventional microscopes. Commercial systems appeared in the 1980s and, despite their high cost, the world market for them is probably between 500 and 1000 instruments per annum, mainly because of their use in biomedical research in conjunction with fluorescent labelling methods. There are many books and review articles on this subject ( e.g. Pawley ( 2006) , Matsumoto( 2002), Wilson (1990) ). The purpose of this chapter is to provide an introduction to optical and engineering aspects that may be o f interest to biomedical users of confocal microscopy. Flying-spot Microscopes A confocal microscope is a special type of ‘flying spot’ microscope. Flying spot systems were developed in the 1950s by combining conventional microscopes with electronics from TV and military equipment. -
Imaging with Second-Harmonic Generation Nanoparticles
1 Imaging with Second-Harmonic Generation Nanoparticles Thesis by Chia-Lung Hsieh In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2011 (Defended March 16, 2011) ii © 2011 Chia-Lung Hsieh All Rights Reserved iii Publications contained within this thesis: 1. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging," Opt. Express 17, 2880–2891 (2009). 2. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes," Biomaterials 31, 2272–2277 (2010). 3. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Second harmonic generation from nanocrystals under linearly and circularly polarized excitations," Opt. Express 18, 11917–11932 (2010). 4. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Digital phase conjugation of second harmonic radiation emitted by nanoparticles in turbid media," Opt. Express 18, 12283–12290 (2010). 5. C. L. Hsieh, Y. Pu, R. Grange, G. Laporte, and D. Psaltis, "Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle," Opt. Express 18, 20723–20731 (2010). iv Acknowledgements During my five-year Ph.D. studies, I have thought a lot about science and life, but I have never thought of the moment of writing the acknowledgements of my thesis. At this moment, after finishing writing six chapters of my thesis, I realize the acknowledgment is probably one of the most difficult parts for me to complete. -
Super-Resolution Imaging by Dielectric Superlenses: Tio2 Metamaterial Superlens Versus Batio3 Superlens
hv photonics Article Super-Resolution Imaging by Dielectric Superlenses: TiO2 Metamaterial Superlens versus BaTiO3 Superlens Rakesh Dhama, Bing Yan, Cristiano Palego and Zengbo Wang * School of Computer Science and Electronic Engineering, Bangor University, Bangor LL57 1UT, UK; [email protected] (R.D.); [email protected] (B.Y.); [email protected] (C.P.) * Correspondence: [email protected] Abstract: All-dielectric superlens made from micro and nano particles has emerged as a simple yet effective solution to label-free, super-resolution imaging. High-index BaTiO3 Glass (BTG) mi- crospheres are among the most widely used dielectric superlenses today but could potentially be replaced by a new class of TiO2 metamaterial (meta-TiO2) superlens made of TiO2 nanoparticles. In this work, we designed and fabricated TiO2 metamaterial superlens in full-sphere shape for the first time, which resembles BTG microsphere in terms of the physical shape, size, and effective refractive index. Super-resolution imaging performances were compared using the same sample, lighting, and imaging settings. The results show that TiO2 meta-superlens performs consistently better over BTG superlens in terms of imaging contrast, clarity, field of view, and resolution, which was further supported by theoretical simulation. This opens new possibilities in developing more powerful, robust, and reliable super-resolution lens and imaging systems. Keywords: super-resolution imaging; dielectric superlens; label-free imaging; titanium dioxide Citation: Dhama, R.; Yan, B.; Palego, 1. Introduction C.; Wang, Z. Super-Resolution The optical microscope is the most common imaging tool known for its simple de- Imaging by Dielectric Superlenses: sign, low cost, and great flexibility. -
NNT '09 Program
NNT '09 Program 9-Nov Wednesday, November 11 12:00 Exhibit set-up, Room J2/J3, San Jose Convention Center 12:00 Registration 17:00 Welcome Reception and Equipment Exhibit, Room J2/J3, San Jose Convention Center Thursday, November 12, Room J1/J4 , San Jose Convention Center Plenary Session - Session Chairs: S. Chou (Princeton) & L. Montelius (Lund) 8:15 Welcome: Stephen Chou and Christie Marrian 8:30 Plenary NNT is Losing the Propaganda War Fabian Pease Stanford University 9:00 Invited 1 Template Infrastructure for Nanoimprint Lithography Nobuhito Toyama Dai Nippon Printing 9:20 Invited 2 NaPANIL: Consolidation of Nanoimprinting for Production Jouni Ahopelto VTT Microsystems and Nanoelectronics 9:40 Invited 3 Shrink-Induced Nanostructures Michelle Khine University of California, Irvine 10:00 Break Magnetics/Biology/Solar - Session Chairs: G. Willson (U. Texas) & H. Schift (PSI) 10:30 Invited 4 The Nano-imprinting Process towards Patterned Media Manufacturing Tsai-Wei Wu HGST 10:50 c1 Large Scale Fabrication of Nanoimprinted Magnetic Nanoparticles with Self-Assembled Templates Wei Hu Stanford University 11:05 c2 Photocatalytic Nanolithography: An Emergent Patterning Technique Relevant to Biotechnology Jane P Bearinger LLNL 11:20 c3 Fabrication of 3D Cell Containers with Integrated Topography by Combined Microscale Arne Schleunitz Paul Scherrer Institut Thermoforming and Thermal Nanoimprint 11:35 c4 Nanoimprinting of Subphthalocyanines for Photovoltaic Applications Xiaogan Liang Lawrence Berkeley National Laboratory 11:50 c5 Nanopatterned anode for organic solar cell by nanoimprint Dae-Geun Choi Korea Institute of Machinery & Materials 12:05 Lunch Electronics/Optoelectronics - Session Chairs: J. Randall (Zyvex) and S. Matsui (Hyogo) 13:30 Invited 5 Nanoimprint lithography for organic thin film transistors Barbara Stadlober Joanneum Research 13:50 c6 Fabrication of organic TFT arrays on an A4-sized flexible sheet using microcontact printing Hiroshi Fujita Dai Nippon Printing Co., Ltd. -
Appendix 1: Confocal Microscopes
Appendix 1: Confocal Microscopes range of confocal microscopes is available - from the highly versatile but very expensive "top end" A instruments, to specialised high-speed imaging and even miniaturised medical instruments. Confocal microscopes are at the forefront of the upsurge in interest in light microscopy in the past 15 years. The technology is still evolving fast - so you should expect major changes and innovations in the coming years. This appendix gives an overview of the hardware for most of the laser scanning confocal microscopes currently available. Although not all manufacturers are described in as much detail as others, this does not in any way reflect on the instruments - this imbalance simply reflects the difficulties in compiling the necessary information for each of the instruments in the time available. I hope that this appendix is an evolving project that will include not only updated, but also expanded information on each ofthe instruments in subsequent editions ofthis book. Laser Spot Scanning Confocal Microscopes These instruments are based on the use of a finely focussed laser "spot" that is scanned across the sampie. The retuming fluorescent or backscattered light is directed through a confocal iris or pinhole that eliminates out-of-focus light. BioRad Cell Science Division _________--'pO<:a..,g.."ec..::3=56 CarIZeiss___________________ p~a~gce~3~8~2 Leica Microsystems_____________ ---"p~a!.llg~e..:!:4=02 Nikon________________________________________ ~p~aAg~e~4~15 Olympus___________________ ~p~a~gce~4~2~O Nipkow Spinning Disk Confocal Microscopes The Nipkow disk contains a large number of fine "pinholes" arranged in a spiral array such that a confocal image is created when the disk is spinning.