Introduction to Confocal Laser Scanning Microscopy

Total Page:16

File Type:pdf, Size:1020Kb

Introduction to Confocal Laser Scanning Microscopy INTRODUCTION TO CONFOCAL MICROSCOPY Matt Renshaw October 2018 The CALM lectures ■ 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 ■ 24th Oct Intro to Confocal Microscopy (Matt Renshaw) Auditorium 1 @10:00 ■ 31st Oct Intro to Live Cell Imaging (Deborah Aubyn) Seminar room 4 @10:00 ■ 7th Nov Intro to Optical Sectioning (Donald Bell) Seminar room 3 & 4 @11:00 ■ 14th Nov Intro to Light Sheet Microscopy (Alessandro Ciccarelli) Seminar room 4 & 5 @10:00 28th Nov Intro to Multiphoton Microscopy (Rocco D’Antuono) Seminar room 4 & 5 at 15:00 Introduction to confocal microscopy Widefield Confocal (yes, both are GFP z stacks) widefield confocal 1 0.8 wf 0.6 cf 0.4 normalised intensity 0.2 0 0 6 11 17 22 distance (microns) Take home message Confocal microscopy improves the optical resolution and contrast of your images by reducing out of focus light Talk outline… ■ Basics of fluorescence microscopy and the principle of confocal microscopy ■ Multi-dimensional image acquisition with a point scanning confocal microscope ■ Advanced applications Jablonski diagram Stokes shift Epifluorescence light path www.leica-microsystems.com/science-lab/fluorescence-in-microscopy/ svi.nl/FluorescenceMicroscope Microscopy is: • creating a magnified, blurred image of your object of interest. Components of blur… 1. Point Spread Function How does the microscope image an infinitely small point of light? Sub-resolution bead Single point of light Airy Disk Light waves interfere and converge on the focal point, creating a diffraction pattern called the Airy Disk. Separating objects – the Airy Disk Rayleigh limit is when the central maxima of one Airy Disk is in line with the first minima of the second Airy Disk Size of the Airy Disk determines minimum resolvable distance of 2 objects http://olympus.magnet.fsu.edu/ Size of the Airy Disk determines minimum resolvable distance of 2 objects Sub-resolution bead in 3D XY YZ Focal plane YZ Focal plane Point Spread Function: the 3D diffraction pattern X Z microscopy.fsu.edu detector Sources of blur pinhole excludes out of focus light, creating an optical section 1. Point Spread Function 2. Light from out of focus areas pinhole X Z excitation light focal sample plane Pinhole blocks diffraction rings Pinhole y x Optical section at resolution limit Pinhole increases contrast by excluding light from the diffraction rings Does the pinhole increase resolution? Laser scanning confocal Widefield Laser illumination scanning Image is built up pixel by pixel Emission light is descanned = focussed onto a static position No pinhole Pinhole excludes out of focus light 10 µm Numerical Aperture ■ Depth of focus ■ PSF ■ size of the airy disk are all related and defined by the angle of light collected by the objective • Numerical Aperture determines: • how much light from the sample is collected by the objective • resolution • the thickness of sample that appears in focus Measured Width of 0.175µ Bead Resolution of 0.175µ Bead Pair 1.1 1.1 1.0 1.0 0.9 0.9 0.8 1.4 NA 0.8 1.4 NA 0.7 0.7 210 nm 0.6 0.6 0.5 0.5 0.4 0.4 0.7 NA 0.7 NA 0.3 0.3 Normalized Intensity Normalized 570 nm Intensity Normalized 0.2 0.2 0.1 0.1 0.0 0.0 0.00 0.15 0.29 0.44 0.59 0.74 0.88 1.03 0.07 0.22 0.37 0.51 0.66 0.81 0.96 1.10 -1.18 -1.03 -0.88 -0.74 -0.59 -0.44 -0.29 -0.15 -1.10 -0.96 -0.81 -0.66 -0.51 -0.37 -0.22 -0.07 Microns Microns ■ Numerical aperture NA = n sin α 1.22 • ■ Resolution d = 2• NA n• ■ Depth of focus D NA2 Optical Sectioning: Confocal Microscopy Other optical sectioning techniques are available. See: Intro to Optical Sectioning talk Talk outline… ■ Basics of fluorescence microscopy and the principle of confocal microscopy ■ Multi-dimensional image acquisition with a point scanning confocal microscope ■ Advanced applications The Tetrahedron of Frustration Standard applications ■ Although there are subtle differences, all of our confocals have these basic functions ■ Software varies, but settings are there somewhere… Excitation and Emission Excitation lasers Detection ■ Adaptable wavelength selection ■ Simultaneous or sequential detection Brief comparison of detectors www.leica-microsystems.com/science-lab/ Optimising acquisition settings ■ Laser power ■ Gain ■ Number of pixels ■ Scanning speed ■ Size of scanning area ■ Averaging Increase laser power = increase signal Increase laser power = increase photo damage Increase laser power = increase signal Increase laser power = increase photo damage Increase gain = increase signal Increase gain = increase noise Increase gain = increase signal Increase gain = increase noise Finding a compromise Finding a compromise Noise can be negative, addition of offset prevents false zeros Finding a compromise Noise can be negative, addition of offset prevents false zeros Achieving resolution: number of pixels 512 x 512 ~500µm per pixel 1024 x 1024 ~250µm per pixel More pixels, more time Achieving resolution: number of pixels 512 x 512 ~500µm per pixel 2048 x 2048 ~125µm per pixel More pixels, more time Achieving resolution: optical zoom 1024 x 1024 ~250µm per pixel 1024 x 1024 ~125µm per pixel Zooming reduces size of scanned area Line averaging reduces noise, but takes longer Averaging 1 Line averaging reduces noise, but takes longer Averaging 4 Line averaging reduces noise, but takes longer Averaging 8 Reduce scanning speed, reduce noise Reduce scanning speed, reduce noise Imaging 3D volumes www.thorlabs.com Imaging deep, increases light scattering Laser power and gain can be adjusted through z stack to compensate Talk outline… ■ Basics of fluorescence microscopy and the principle of confocal microscopy ■ Multi-dimensional image acquisition with a point scanning confocal microscope ■ Advanced applications Nikon W1 spinning disk confocal ■ Speed ■ Reduced photo-toxicity ■ Photo manipulation Resonant scanning on the Olympus FV3000 ■ rapidly oscillating resonant mirror scanners ■ Very fast frame speeds (~400 per second) ■ Permits very low laser power ■ Combined with rolling averaging (post processing) can detect high quality images of dynamic processes Zeiss LSM 880 with Airyscan Pinhole diameter, resolution and transmission 0.2 AU = 1.4 x improvement 1.1 100 1.0 80 0.9 60 0.8 40 Resolution 20 0.7 5. Pinhole transmission % Pinhole transmission 0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 0.5 1.0 1.5 2.0 2.5 3.0 4.0 4.5 5.0 Pinhole diameter (AU) Pinhole diameter (AU) Airyscan detector is 32x 0.2 AU mini GaAsP detectors 0.2 AU Joseph Huff Nature Methods volume12, page1205 (2015) Conventional Airyscan Alex van Vliet, Tooze Lab Spectral imaging and unmixing ■ separating fluorophores with close emission spectra e.g. Alexa fluor 555 and Mito Tracker orange Relative intensity (%) intensity Relative Wavelength (nm) Whole spectrum of emitted light collected by spectral detector (ChS) http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html 8.7 nm intensity bins 415 nm 686 nm Alexa 555 labelled microtubules and Mito tracker orange mitochondria unmixing Multiphoton ■ Multiphoton laser allows very deep tissue imaging ■ See Introduction to Multiphoton Imaging talk (Rocco, 28th Nov) ■ MP lasers can also be used for cutting experiments MP – ablation experiments Sophie Herszterg, Vincent lab Fluorescent Recovery After Photobleaching ■ Bleach region of interest ■ Measure recovery of fluorescence Using FRAP to investigate cell investigate to FRAP Using - cell cell interactions 180815 CAP cKO +CAP 14 180815 CAP cKO 7 ) ) h h c c a a Alex Hunt, Hunt, Alex e e l l 110 110 b b - - 1 1 e e 100 100 r r p p 90 90 2 2 o o t t 80 80 e e y y 3 70 3 t 70 v t v i i i i t t s s 60 60 a a l n l n 4 4 e e e e 50 50 t r t r n n e I e I 40 5 40 5 g g a a 30 30 t t Treeck 6 6 n n 20 20 e e c c 10 7 10 7 r r e e 0 0 p p 8 8 . 0 30 60 90 120 0 30 60 90 120 u u . a a ( ( Time (s) Time (s) lab New Leica SP8 FALCON Time-resolved, single-photon detection 460 700 Pulsed, white-light laser Fluorescence Lifetime Imaging (FLIM) GFP mCherry ■ Quantify FRET ■ Detect protein interactions Mean tau = 2320 ps 2658 ps Roman Fedoryshchak, Treisman lab Thank you for your attention! Good sources of info… (Google is always useful too) ■ https://www.leica-microsystems.com/science-lab/ ■ https://www.microscopyu.com/ ■ http://olympus.magnet.fsu.edu/index.html ■ http://zeiss-campus.magnet.fsu.edu/ ■ http://micro.magnet.fsu.edu/ ■ https://svi.nl/ ■ Imaging Helpdesk (twice monthly in cafe) .
Recommended publications
  • 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.
    [Show full text]
  • Multiphoton Microscopy
    Living up to Life Multiphoton Microscopy 1 Jablonski Diagram: Living up to Life Nonlinear Optical Microscopy F.- Helmchen, W. Denk, Deep tissue two-photon microscopy, Nat. Methods 2, 932-940 2 Typical Samples – Living up to Life Small Dimensions & Highly Scattering • Somata 10-30 µm • Dendrites 1-5 µm • Spines ~0.5 µm • Axons 1-2 µm ls ~ 50-100 µm (@ 630 nm) ls ~ 200 µm (@ 800 nm) T. Nevian Institute of Physiology University of Bern, Switzerland F.- Helmchen, W. Denk Deep tissue two-photon microscopy. Nat. Methods 2, 932-940 3 Why Multiphoton microscopy? Living up to Life • Today main challenge: To go deeper into samples for improved studies of cells, organs or tissues, live animals Less photodamage, i.e. less bleaching and phototoxicity • Why is it possible? Due to the reduced absorption and scattering of the excitation light 4 The depth limit Living up to Life • Achievable depth: ~ 300 – 600 µm • Maximum imaging depth depends on: – Available laser power – Scattering mean-free-path – Tissue properties • Density properties • Microvasculature organization • Cell-body arrangement • Collagen / myelin content – Specimen age – Collection efficiency Acute mouse brain sections containing YFP neurons,maximum projection, Z stack: 233 m Courtesy: Dr Feng Zhang, Deisseroth laboratory, Stanford University, USA Page 5 What is Two‐Photon Microscopy? Living up to Life A 3-dimensional imaging technique in which 2 photons are used to excite fluorescence emission exciting photon emitted photon S1 Simultaneous absorption of 2 longer wavelength photons to
    [Show full text]
  • Adaptive Optics in Microscopy
    Downloaded from http://rsta.royalsocietypublishing.org/ on February 3, 2015 Phil. Trans. R. Soc. A (2007) 365, 2829–2843 doi:10.1098/rsta.2007.0013 Published online 13 September 2007 Adaptive optics in microscopy BY MARTIN J. BOOTH* Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK The imaging properties of optical microscopes are often compromised by aberrations that reduce image resolution and contrast. Adaptive optics technology has been employed in various systems to correct these aberrations and restore performance. This has required various departures from the traditional adaptive optics schemes that are used in astronomy. This review discusses the sources of aberrations, their effects and their correction with adaptive optics, particularly in confocal and two-photon microscopes. Different methods of wavefront sensing, indirect aberration measurement and aberration correction devices are discussed. Applications of adaptive optics in the related areas of optical data storage, optical tweezers and micro/nanofabrication are also reviewed. Keywords: adaptive optics; aberrations; confocal microscopy; multiphoton microscopy; optical data storage; optical tweezers 1. Introduction Optical microscopes are essential tools in many scientific fields. In the life sciences, they are widely used for the visualization of cellular structures and sub- cellular processes. Confocal and multiphoton microscopes are particularly important in this respect as they produce three-dimensional images of volumetric objects. However, the resolution of these microscopes is often adversely affected by the optical properties of the specimen itself. Spatial variations in the refractive index of the specimen introduce optical aberrations that compromise image quality. This is a particular problem when imaging deep into thick biological specimens.
    [Show full text]
  • 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.
    [Show full text]
  • Electron Microscopy and the Investigation of New Infectious Diseases
    Review Electron microscopy and the investigation of new infectious diseases Alan Curry@) Objectives: To review and assess the role of electron microscopy in the investigation of new infectious diseases. Design: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. Results: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. Conclusions: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases. Int J Infect Dis 2003; 7: 251-258 INTRODUCTION at individually by a skilled microscopist have con- The electron microscope was developed just before tributed to the decline of electron microscopy. Against World War II in several countries, but particularly in this background, the inevitable question must be Germany.l The dramatic increase in resolution available asked-does electron microscopy still have a useful in comparison with light microscopy promised to role to play in the investigation of emerging or new revolutionize many aspects of cell biology, virology, infectious diseases? bacteriology, mycology and protozoan parasitology.
    [Show full text]
  • About Resolution
    pco.knowledge base ABOUT RESOLUTION The resolution of an image sensor describes the total number of pixel which can be used to detect an image. From the standpoint of the image sensor it is sufficient to count the number and describe it usually as product of the horizontal number of pixel times the vertical number of pixel which give the total number of pixel, for example: 2 Image Sensor & Camera Starting with the image sensor in a camera system, then usually the so called modulation transfer func- Or take as an example tion (MTF) is used to describe the ability of the camera the sCMOS image sensor CIS2521: system to resolve fine structures. It is a variant of the optical transfer function1 (OTF) which mathematically 2560 describes how the system handles the optical infor- mation or the contrast of the scene and transfers it That is the usual information found in technical data onto the image sensor and then into a digital informa- sheets and camera advertisements, but the question tion in the computer. The resolution ability depends on arises on “what is the impact or benefit for a camera one side on the number and size of the pixel. user”? 1 Benefit For A Camera User It can be assumed that an image sensor or a camera system with an image sensor generally is applied to detect images, and therefore the question is about the influence of the resolution on the image quality. First, if the resolution is higher, more information is obtained, and larger data files are a consequence.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • Applications of Microscopy in Bacteriology
    Microscopy Research, 2016, 4, 1-9 Published Online January 2016 in SciRes. http://www.scirp.org/journal/mr http://dx.doi.org/10.4236/mr.2016.41001 Applications of Microscopy in Bacteriology Mini Mishra1, Pratima Chauhan2* 1Centre of Environmental Studies, Department of Botany, University of Allahabad, Allahabad, India 2Department of Physics, University of Allahabad, Allahabad, India Received 28 September 2015; accepted 2 January 2016; published 5 January 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract Bacteria are smallest primitive, simple, unicellular, prokaryotic and microscopic organisms. But these organisms cannot be studied with naked eyes because of their minute structure. Therefore in search for the information about the structure and composition of bacterial cells, cell biologist used light microscopes with a numerical aperture of 1.4 and using wavelength of 0.4 µm separa- tion. But there are still certain cellular structures that cannot be seen through naked eyes, and for them electron microscope is used. There are certain improved types of light microscope which can be incorporated to improve their resolving power. Hence microscopy is playing a crucial role in the field of bacteriology. Keywords AFM, SEM, TEM, Microscopy, Bacteriology 1. Introduction To get acquainted with the world of bacteria like small organisms, very effective and advanced technique is re- quired. The size of bacteria ranges between 0.5 - 5.0 micrometer in length; the smallest of them are members of mycoplasma which measures 0.3 micrometers [1].
    [Show full text]
  • High-Aperture Optical Microscopy Methods for Super-Resolution Deep Imaging and Quantitative Phase Imaging by Jeongmin Kim a Diss
    High-Aperture Optical Microscopy Methods for Super-Resolution Deep Imaging and Quantitative Phase Imaging by Jeongmin Kim A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Engineering { Mechanical Engineering and the Designated Emphasis in Nanoscale Science and Engineering in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Xiang Zhang, Chair Professor Liwei Lin Professor Laura Waller Summer 2016 High-Aperture Optical Microscopy Methods for Super-Resolution Deep Imaging and Quantitative Phase Imaging Copyright 2016 by Jeongmin Kim 1 Abstract High-Aperture Optical Microscopy Methods for Super-Resolution Deep Imaging and Quantitative Phase Imaging by Jeongmin Kim Doctor of Philosophy in Engineering { Mechanical Engineering and the Designated Emphasis in Nanoscale Science and Engineering University of California, Berkeley Professor Xiang Zhang, Chair Optical microscopy, thanks to the noninvasive nature of its measurement, takes a crucial role across science and engineering, and is particularly important in biological and medical fields. To meet ever increasing needs on its capability for advanced scientific research, even more diverse microscopic imaging techniques and their upgraded versions have been inten- sively developed over the past two decades. However, advanced microscopy development faces major challenges including super-resolution (beating the diffraction limit), imaging penetration depth, imaging speed, and label-free imaging. This dissertation aims to study high numerical aperture (NA) imaging methods proposed to tackle these imaging challenges. The dissertation first details advanced optical imaging theory needed to analyze the proposed high NA imaging methods. Starting from the classical scalar theory of optical diffraction and (partially coherent) image formation, the rigorous vectorial theory that han- dles the vector nature of light, i.e., polarization, is introduced.
    [Show full text]
  • The Development of High Performance Liquid
    Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 3-23-2010 The evelopmeD nt of High Performance Liquid Chromatography Systems for the Analysis of Improvised Explosives Megan N. Bottegal Florida International University, [email protected] DOI: 10.25148/etd.FI10041603 Follow this and additional works at: https://digitalcommons.fiu.edu/etd Recommended Citation Bottegal, Megan N., "The eD velopment of High Performance Liquid Chromatography Systems for the Analysis of Improvised Explosives" (2010). FIU Electronic Theses and Dissertations. 154. https://digitalcommons.fiu.edu/etd/154 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida THE DEVELOPMENT OF OPTIMIZED HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SYSTEMS FOR THE ANALYSIS OF IMPROVISED EXPLOSIVES A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in CHEMISTRY by Megan Nicole Bottegal 2010 To: Dean Kenneth Furton College of Arts and Sciences This dissertation, written by Megan Nicole Bottegal, and entitled The Development of Optimized High Performance Liquid Chromatography Systems for the Anlysis of Improvised Explosives, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. ____________________________________ Jose Almirall ____________________________________ John Berry ____________________________________ William Hearn ____________________________________ Fenfei Leng ____________________________________ DeEtta Mills ____________________________________ Bruce McCord, Major Professor Date of Defense: March 23, 2010 The dissertation of Megan Nicole Bottegal is approved.
    [Show full text]
  • Contrast Enhancement for Topographic Imaging in Confocal Laser Scanning Microscopy
    applied sciences Article Contrast Enhancement for Topographic Imaging in Confocal Laser Scanning Microscopy Lena Schnitzler *,†, Markus Finkeldey †, Martin R. Hofmann and Nils C. Gerhardt Photonics and Terahertz Technology, Ruhr University Bochum, 44780 Bochum, Germany * Correspondence: [email protected] † These authors contributed equally to this work. Received: 26 June 2019; Accepted: 29 July 2019; Published: 31 July 2019 Featured Application: The method for contrast enhancement presented in this work, can be used to increase the image contrast of topographic structures. It might be an essential tool for non-destructive testing in the quality management of microcontroller production, in the processing of semiconductors or in the developement and characterization of MEMs. Even structures with low height variance can be observed. Abstract: The influence of the axial pinhole position in a confocal microscope in terms of the contrast of the image is analyzed. The pinhole displacement method is introduced which allows to increase the contrast for topographic imaging. To demonstrate this approach, the simulated data of a confocal setup as well as experimental data is shown. The simulated data is verified experimentally by a custom stage scanning reflective microscopy setup using a semiconductor test target with low contrast structures of sizes between 200 nm and 500 nm. With the introduced technique, we are able to achieve a contrast enhancement of up to 80% without loosing diffraction limited resolution. We do not add additional components to the setup, thus our concept is applicable for all types of confocal microscopes. Furthermore, we show the application of the contrast enhancement in imaging integrated circuits. Keywords: microscopy; confocal microscopy; contrast enhancement 1.
    [Show full text]