Characterization of Fluorophores for Two-Photon and STED Microscopy in Tissue Master’s Thesis MSc in Nanobioscience and Interdisciplinary Science Studies Authored by Bjarne Thorsted ∗ Supervisor Jonathan R. Brewer Memphys - Center for Biomembrane Physics Department of Physics, Chemistry & Pharmacy University of Southern Denmark February 2015 ∗[email protected] i Abstract Two-Photon Excitation Microscopy (TPEM) uses high-intensity long- wavelength laser light to excite fluorophores and provide resolution compa- rable to confocal microscopy with an inherent optical sectioning and deep tissue imaging ability. Stimulated Emission Depletion (STED) use a deex- citation laser to reduce the area of excited fluorophores, which practically results in an increased resolution of down to 20 nm. These two microscopy techniques have a large potential if combined, because of their individual advantages. Characterizing fluorophores for use in these systems is therefore of great interest to the scientific community. In this thesis, I present the results of my efforts to successfully build a two-photon spectrofluorometer capable of automatic acquisition of spectral data of commercial and novel dyes, in the form of two-photon absorption cross sections. I also show that it is possible to use the two commercial dyes Abberior Star 440SX and Abbe- rior Star 488 to image specimens in TPEM by exciting them at 860 nm and 1010 nm, respectively. I elaborate on the suitability of the dyes in regards to TPEM and provide data from in situ experiments with some of the dyes in human skin tissue imaged by confocal, TPE and STED microscopy that support earlier reports of the spatial differentiation between two proteins, claudin-1 and desmoglein-1, involved in maintaining the skin barrier, showing that claudin-1 is virtually non-present in the stratum corneum and mainly located in the stratum granulosum, whereas desmoglein-1 is found throughout the epidermis. iii Acknowledgments This project has been part of my everyday life for more than a year now. Through the ups and downs, I have always been deeply grateful for the opportunity to work on this project, which has given me so many great experiences. My supervisor, Jonathan Brewer, has been both a mentor and a friend to me during this project. I have truly learned a lot from him. He has been invaluable to me in many ways, and I cannot thank him enough for his support and high spirits in even the bleakest of situations. I would also like to thank Jes Dreier for helping me use the Huygens image software and Till Leißner for helping maintain and improve upon the microscope setup. To all my friends, I owe a great thanks. I could not have made it through without you. Henrik Thoke, in particular, has always been great at asking the right questions when my own mind got stuck in a rut, and Anne Zebitz has never failed to help me get out of my cave and relax a little on a saturday night. Finally, I would like to thank my family for always being supportive of my life decisions, and my lovely niece, Sarah, who means the world to me and to whom I dedicate this work. Contents Glossary vii 1 Introduction 1 2 Theory 5 2.1 Fluorescence.................................... 5 2.2 Two-Photon Absorption............................ 8 2.2.1 One-Photon Absorption Cross Section............. 11 2.2.2 Two-Photon Absorption Cross Section............. 14 2.3 Stimulated Emission............................... 19 2.4 Confocal Microscopy.............................. 20 2.4.1 Optical Resolution.......................... 23 2.5 STED Microscopy................................ 25 2.6 Cell Junctions................................... 30 2.6.1 Desmosomes.............................. 31 2.6.2 Tight Junctions............................. 32 3 Materials and Methods 33 3.1 Materials...................................... 33 3.2 Software and Equipment............................ 35 3.2.1 Automation Scripts.......................... 37 autoAOTF.bsh............................. 37 auto.VBS................................. 37 3.3 2-photon Absorption Cross-section Measurements........... 38 3.3.1 Data Acquisition............................ 38 3.3.2 Data Analysis.............................. 39 twopa.m................................. 39 3.4 Sample Preparation............................... 39 v vi Contents 3.5 STED Microscopy................................ 40 4 Results and Discussion 41 4.1 Two-Photon Absorption Cross Sections.................. 41 4.2 STED Microscopy................................ 45 4.2.1 Photostability.............................. 52 5 Conclusion and Perspective 55 Bibliography 59 A Appendix A-1 A.1 Mowiol Recipe .................................. A-1 B 2PA Cross Sectional DataB- 1 C Lab Automation and Matlab ScriptsC- 1 C.1 Automation Control for the Equipment..................C- 1 C.1.1 autoAOTF.bsh.............................C- 1 C.1.2 auto.VBS.................................C- 10 C.1.3 background.VBS............................C- 12 C.1.4 Free-run.VBS..............................C- 13 C.1.5 settings.VBS...............................C- 14 C.2 MATLAB® Scripts................................C- 15 C.2.1 concentration.m............................C- 15 C.2.2 twopa.m.................................C- 16 C.2.3 twopacollect.m.............................C- 23 Glossary c.c The complex conjugate of a number or expression. A complex conjugate is identical to its partner except with opposite sign of the imaginary part. The complex conjugate of z = a + i b is z¯ = a i b − CLSM Confocal Laser Scanning Microscope. Dirac delta function A function defined such that it is has a value of zero at all point except zero, and the area under the line is equal to 1. It is mathematically defined as if x = 0 1 δ(x) = 1 δ(x) = 1. ¨0 if x = 0 6 Z−∞ It is related to the Kronecker delta. ~h The reduced Plank’s constant, equal to Planck’s constant h divided by 2π, or 34 1.054571726(47) 10− Js × i 2 The imaginary unit, defined such that i = 1. Complex numbers have a real and imaginary part and can be represented− in the complex plane as a number z = a + i b, where a is the real part and b is the imaginary part with the unit i vii viii Glossary Kronecker delta Is a function of two (or more) variables. If the variables are equal, the function is 1 and otherwise 0. Mathematically, this is expressed as 1 if i = j δi j = . 0 if i = j ¨ 6 An identity matrix has the same properties as the Kronecker delta λ 1) The wavelength of light, usually measured in nanometer (nm). 2) An expansion parameter used in perturbation theory. NA Numerical aperture of a lens or objective. It is defined as NA = n sinα, where n is the refractive index and α is the angular aperture NIR Near-infrared; a range in the electromagnetic field starting at 700 nm and extending up to about 1400 nm, depending on the standard definition used ν Frequency of a photon. Often used to denote the energy since this is propor- tional to the frequency by E = hν. ! Angular frequency. Point spread function A response function, that describes the spatial distribution of light from a point-like source or object. The ideal description being an Airy function ~r The position vector of an object. In a cartesian coordinate system, the vector consists of the three scalars (x, y, z). (N) σi f The Nth order absorption cross section of a material from state i to f . The order indicates the number of simultaneous photons involved in the absorption process Glossary ix STED A technique for resolving objects smaller than the diffraction limit typically allows for.. t A variable denoting time. TEM Transmission Electron Microscopy. TPE Two-Photon Excitation. TPEM Two-Photon Excitation Microscopy. CHAPTER 1 Introduction Bioimaging has been a fundamental tool for several decades in the fields of histology, and cell and molecular biology. Especially, the ability to do specific labeling and imaging of different proteins, membranes, and organelles with the fluorescence- based microscopy technique has advanced the knowledge in these fields tremen- dously [1]. Optical microscopy has enjoyed many advances since its invention. Fluorescence microscopy enabled researches to view specific structures and proteins that are normally not visible with plain white light by staining samples with anti-body linked fluorophores that selectively adhere to the sample. Confocal fluorescence microscopy greatly enhanced the definition of micrographs, by filtering out-of- focus light from the sample, albeit at the expense of contrast [2]. This was a small sacrifice, though, since researchers were now able to precisely control in which particular depth they wanted to examine their samples by moving the focal plane up and down in the sample. This also made it possible to build 3D models of the samples by combining images taken at various depths, giving easier access to spatial information. Two-Photon Excitation (TPE) is similar to confocal microscopy, in that both have an inherent ability to image only limited sections of the sample in the plane of focus, enabling researchers to create 3D images of the samples. Where the latter rely on insertion of pinholes in the light path to control the light entering and leaving the sample, the former uses the physical properties of a material response to high intensity laser light [3]. Two-Photon Excitation Microscopy (TPEM) utilize longer wavelength light to excite the fluorophores in a sample, which results in a decrease in resolution, but offers other advantages to make up for it. Because thick tissue samples scatters light more than a thin cell culture sample, the high intensity light used in TPEM generally provides better contrast than does its confocal counterpart. TPEM also offers superior depth penetration over confocal microscopy, in part because the longer wavelengths are less prone to scattering in biological tissue, but 1 Characterization of Fluorophores for 2P & STED Microscopy . also because of the high intensity used to illuminate the sample. Where confocal microscopy is ineffective beyond a few hundred micrometers, its two-photon cousin can image up to one millimeter into a sample [4].