DOCSLIB.ORG

STED Microscopy Sample Preparation Dr

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
STED Microscopy Sample Preparation Dr STED Microscopy Sample preparation Dr. Nathalie Garin Components of a microscope Objective lens Cover slip Immersion Sample The last 3 optical elements are usually added by the user ! Coverslip thickness . Objective lenses designed for use with 170 µm coverslips (i.e. considered in the optical calculation of the lens) . Some lenses are equipped with an adjustable collar correcting for different thickness . Mismatch causes o spherical and chromatic aberration o spread of focus spot o loss in resolution o intensity decreases o distortion of structures inside deeper layers of the sample 15 µm cover slip deviation approx. deteriorates ½ of resolution & intensity Coverslip Thickness # Thickness [mm] 0 0,08 – 0,12 1 0,13 – 0,16 1,5 0,16 – 0,19 2 0,19 – 0,23 Source: Hecht-Assistent / Menzel Coverslip Sources www.leica-microsystems.com/products/total- histology/consumables/catalog/catalog-us/ http://www.hecht-assistent.de/index-us.html http://www.menzel.de/ APPLICATIONS Protein localization and dual color imaging STED Microscopy – sample preparation 6 Nuclear proteins labelled with Alexa 488 D. Gruenwald; J. Hopkins, New York direct immunofluorescence STED CW: Spectral Conditions Example: OregonGreen 488 No Excitation at lSTED Dyes with excitation from 458 nm to 514 nm Depletion at 592 nm STED: Spectral Conditions Excitation STED Detection Band No Excitation at lSTED Dyes with excitation at 532 nm or 640 nm Tunable depletion from 660 to 780 nm Some working green dyes for CW STED Dye Vendor Laser line 2 color Alexa 488 Invitrogen 488 or 514 Atto 488 ATTO-TEC 488 or 514 Chromeo 488 Active Motif 488 or 514 Chromeo 505 Active Motif 488 or 514 recommended DyLight 488 Pierce Technology 488 or 514 FITC Invitrogen 488 or 514 FluoProbes 488 Interchim 488 or 514 Oregon Green 488 Invitrogen 488 or 514 recommended Oregon Green 514 Invitrogen 514 STED Microscopy – Principles and Applications 10 Some working dyes for pulsed STED Dye Vendor Laser lines 2 color Abberior STAR 635 Abberior 640/750 recommended Alexa 647 Invitrogen 640/750 Atto 647N ATTO-TEC 640/750 recommended Atto 655 ATTO-TEC 640/750 Atto 665 ATTO-TEC 640/775 STED Microscopy – Principles and Applications 11 2 color STED images of neurons courtesy Dr. W. Zuschratter, IfN, Magdeburg STED Microscopy – Principles and Applications 13 2 color STED: BD Horizon V500 Oregon Green 488 Channel 1 Channel 2 Multicolor Imaging – TCS STED CW Fluorophore Exc / nm Em / nm Source Coupling NBD-X 467 538 Invitrogen Succinimidyl Pacific Orange 400 556 Invitrogen IgG Horizon V500 415 500 Becton & Dickinson Streptavidin Excitation @ 458 nm Multicolor Imaging Example: Pacific Orange, Alexa488 STED raw data STED color separation Sample courtesy: MPI Goettingen, NBDX & Alexa488 (postprocessing: spectral separation) Some large Stoke’s shift dyes for CW STED Dye Vendor Laser line remarks Abberior STAR 440SX Abberior 458 recommended Atto 425 Rockland 458 BD Horizon V500 Becton Dickinson 458 recommended Pacific Orange Invitrogen 458 STED Microscopy – Principles and Applications 18 Some large Stoke’s shift dyes for pulsed STED Dye Vendor Laser lines 2 color Alexa 532 Invitrogen 531/750 Chromeo 494 Active Motif 531/750 recommended Mega 520 Sigma-Aldrich 531/775 STED Microscopy – Principles and Applications 19 Configuration examples TCS STED CW Dye1 Dye2 Name Excitation Emission Name Excitation Emission BD Horizon V500 458 465 - 515 OregonGreen488 or 514 514 520 – 580 Pacific orange 458 535 - 580 Alexa488 (or any green 488 500 – 580 dye) NBDX 458 500 - 580 Alexa488 (or any green 488 500 – 580 dye) TCS STED (depletion at 765nm) Dye1 Dye2 Name Excitation Emission Name Excitation Emission Chromeo494 531 505 - 605 ATTO647N 635 665 – 705 Mega520 531 665 - 705 ATTO 655 635 665 – 705 Tips and tricks I . Carry out your stainings similar to regular fluorescent imaging. Good samples in regular fluorescence imaging are also suitable for STED imaging. Check the mounting medium for compatibility with the fluorescent dyes (especially concerning the large Stoke’s shift dyes) and doesn’t show high levels of auto-fluorescence. Recommended mounting media are mowiol, 86% glycerol, Prolong Gold and TDE, with or without anti-fade agents. Vectashield isn’t compatible with large Stoke’s shift dyes. Use sequential acquisition for 2 color STED imaging. Labeling procedure and density may influence image quality. Immunolabeling, classical steps . Fixation (usually 4% PAF) . Washing . Permeabilisation (Triton, saponin) . Washing . Blocking (BSA or NGS) . Incubation of the primary antibody (recommended : overnight at 4°C) Immunolabeling, classical steps . Fixation (usually 4% PAF) . Washing . Permeabilisation (Triton, saponin) . Washing . Blocking (BSA or NGS) . Incubation of the primary antibody (recommended : overnight at 4°C) . Washing . Incubation with secondary antibody 1 hour at RT . Washing . Postfixation in 4% PFA . washing Consider the size of the label Y primary Ab dye secondary Ab Y 7 – 10nm Fab fragments: 3.5 – 5.5nm Y Y Y Y Y Y direct IF indirect IF 25 Thickness of sample Refractive Index Match & Mismatch Oil Oil Coverslip Coverslip Oil Water Refractive indMatchex mismatch results in spherical aberrationMismatch Matching RIs crucial for confocal microscopy …and even more so for nanoscopy! A. Egner and S. W. Hell Doughnut visualized with 80 nm gold beads Reflection mode, 488 nm & 592 nm, zoom 64 center section yz xz Oil Objective PL APO 63x1.3 Sample embedded in Glycerol (80/20%) Thick specimen (100µm): muscle fibers Mounting & Immersion Refractive indices Immersion / Mounting Media Good results were obtained with: . Water: 1.33447 . Mowiol . Glycerol 23°C: 1.45100 . Glycerol . Vectashield (100%) 1.46 . ProLongGold . Mowiol 1.46 . 86% glycerol + 4% NPG (N-propyl-gallate) . TDE/Water: 1.33 – 1.52 . 86% glycerol + 2.5% DABCO . Immersion Oil: 1.518 at pH7.4 . Glas 1.52 Penetration depth To avoid: 10 – 25 µm . Vectashield (Vectorlabs) . Slowfade (invitrogen) . Para-phenylenediamine (PPD) Protocoles Mowiol embedding media (pH7.4): •take 6g Glycerol (analytical grade) •add 2.4 g Mowiol 4-88 (Calbiochem # 475904) •add 6 ml Aqua dest. •add 12 ml 0.2 M TRIS Buffer pH74. •add 2.5% DABCO (=Anti -Bleaching-Reagent 1,4- Diazabicyclo-82.2.29-octan; Fluka #33480) •stir for 4 hours (magnetic stirrer) •let rest suspension for 2 hours •incubate for 10 min at 50°C (water bath) •centrifuge at 5000 g for 15 min •take the supernatant and freeze it in aliquots at -20°C Glycerol – PBS- antifading (pH7.4): •86%glycerol+4% n-propyl-gallate(NPG) Thiodiethanol (pH7.4) •TDE, Sigma, #88559) has been used with excellent results (Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW (2007). “2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.” Microsc Res Tech. 70(1):1- 9.). The TDE concentration must be gradually enhanced to obtain a final refractive index of 1.514, which is reached using a TDE concentration of 97%. Sequential steps in TDE 50%, 70% (15 minutes at each step), then in 97% + antifade as final mounting media must be undertaken •If using TDE, the coverslip must be sealed using invisible nail Mounting media must have antifade freshly mixed in it. •N-propyl gallate (NPG, 4%) while not very soluble, is non-toxic and can be used on live cells. •DABCO (2.5%) is a well-known antifade. APPLICATIONS STED live-imaging 32 CW STED imaging with fluorescent proteins proteins vendor laser line remarks eGFP Tsien et al 488 eYFP Invitrogen 514 recommended Citrin Tsien et al 514 recommended Venus Miyawaki et al 514 Potential dyes for STED CW proteins laser line Cerulean 458 CyPet 458 eCFP 458 Emerald 488 mBanana 514 Topaz 514 YPet 514 ZsYellow 514 STED Microscopy – Principles and Applications 35 More live imaging: (Abp1-eGFP in yeast cells) Confocal Courtesy of Marko Kaksonen EMBL, Heidelberg 1 Hz STED Live Cell STED with Fluorogen Activating Proteins (FAPs) Specific labeling of genetically encoded marker without background problems Fluorogen Protein-FAP DNA live cell Szent et al. Nature Biotechnol 2008; Fitzpatrick et al. Bioconjugate Chem 2009 Live Cell STED with Fluorgen Activating Proteins (FAPs) Confocal STED Fluorogen Activating Protein (FAP) labeled ADRB2 TCS STED in living NIH 3T3 cells Images: James Fitzpatrick/Jochen Sieber STED Szent et al. Nature Biotechnol 2008; Fitzpatrick et al. Bioconjugate Chem 2009 Tips and tricks II . Bleaching rate of fluorescent proteins and dyes are directly correlated with the pixel dwell time. Fast scan speed with higher number of averaging are more gentle to the fluorophore and thus may produce a higher number of frames before bleaching. Influence of scan speed Alexa 488 in - Tubulin 4 3 2 1 Resonant bleachingexponents [images] Galvoscanner 0 0 200 400 600 800 1000 1200 1400 8000 scan rate [Hz] Tips and tricks II . Bleaching rate of fluorescent proteins and dyes are directly correlated with the pixel dwell time. Fast scan speed with higher number of averaging are more gentle to the fluorophore and thus may produce a higher number of frames before bleaching. Signal to noise ratio (SNR) is correlated to the pixel size. Bigger pixel give “brighter” images. Oversampling of at least 2 is still recommended . Slightly reduce STED depletion power for better SNR. Compromising the resolution with lower STED intensities may increase SNR. Change the pinhole size. Increasing the pinhole size may increase the SNR in samples that aren’t too densely labeled. Consider the use of lower excitation power and accumulation with low noise detectors (HyD and APD at low gains). Tips and tricks II . Mounting medium: o Antifadings: vitamin C, A, …. o No riboflavin or phenol red Living up to Life confocal STED Thank you for your attention!.
Load more

Recommended publications
  • The Green Fluorescent Protein
    P1: rpk/plb P2: rpk April 30, 1998 11:6 Annual Reviews AR057-17 Annu. Rev. Biochem. 1998. 67:509–44 Copyright c 1998 by Annual Reviews. All rights reserved THE GREEN FLUORESCENT PROTEIN Roger Y. Tsien Howard Hughes Medical Institute; University of California, San Diego; La Jolla, CA 92093-0647 KEY WORDS: Aequorea, mutants, chromophore, bioluminescence, GFP ABSTRACT In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrin- sically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the rela- tion between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories. CONTENTS NATURAL AND SCIENTIFIC HISTORY OF GFP .................................510 Discovery and Major Milestones .............................................510 Occurrence, Relation to Bioluminescence, and Comparison with Other Fluorescent Proteins .....................................511 PRIMARY, SECONDARY, TERTIARY, AND QUATERNARY STRUCTURE ...........512 Primary Sequence from
    [Show full text]
  • Microenvironment-Triggered Dual-Activation of a Photosensitizer
    www.nature.com/scientificreports OPEN Microenvironment‑triggered dual‑activation of a photosensitizer‑ fuorophore conjugate for tumor specifc imaging and photodynamic therapy Chang Wang1, Shengdan Wang1, Yuan Wang1, Honghai Wu1, Kun Bao2, Rong Sheng1* & Xin Li1* Photodynamic therapy is attracting increasing attention, but how to increase its tumor‑specifcity remains a daunting challenge. Herein we report a theranostic probe (azo‑pDT) that integrates pyropheophorbide α as a photosensitizer and a NIR fuorophore for tumor imaging. The two functionalities are linked with a hypoxic‑sensitive azo group. Under normal conditions, both the phototoxicity of the photosensitizer and the fuorescence of the fuorophore are inhibited. While under hypoxic condition, the reductive cleavage of the azo group will restore both functions, leading to tumor specifc fuorescence imaging and phototoxicity. The results showed that azo‑PDT selectively images BEL‑7402 cells under hypoxia, and simultaneously inhibits BEL‑7402 cell proliferation after near‑infrared irradiation under hypoxia, while little efect on BEL‑7402 cell viability was observed under normoxia. These results confrm the feasibility of our design strategy to improve the tumor‑ targeting ability of photodynamic therapy, and presents azo‑pDT probe as a promising dual functional agent. Cancer is one of the most common causes of death, and more and more therapeutic strategies against this fatal disease have emerged in the past few decades. Among these strategies, photodynamic therapy has attracted much attention1. Tis therapy is based on singlet oxygen produced by photosensitizers under the irradiation with light of a specifc wavelength to damage tumor tissues (Fig. 1a). Since the photo-damaging efect is induced by the interaction between a photosensitizer and light, tumor-specifc therapy may be realized by focusing the light to the tumor site.
    [Show full text]
  • Fluorescent Protein-Based Tools for Neuroscience
    !1 !2 Fluorescent protein-based tools Outline for neuroscience An animatd primer on biosensor development Fluorescent proteins (FPs) Robert E. Campbell Department of Chemistry Other fluorophore technologies Single FP-based biosensors Imaging Structure & Function in the Nervous System Cold Spring Harbor, July 31, 2019. Lots of structural Lots of structural information information & Transmitted light Fluorescence microscopy of fluorescent color microscopy of live cells live cells No molecular provides molecular information information (more colors = more information) !5 !6 Fluorescence microscopy requires fluorophores Non-natural fluorophores for protein labelling Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N 495 nm 519 nm 557 nm 576 nm - - CO2 CO2 S O O O N O N C N N C S ϕ = quantum yield - - ε = extinction coefficient CO2 CO2 ϕ Brightness ~ * ε S C i.e., for fluorescein N Proteins of interest ϕ = 0.92 N Fluorescein Tetramethylrhodamine ε = 73,000 M-1cm-1 C S (FITC) (TRITC) A non-natural fluorophore must be chemically linked to Non-natural fluorophores made by chemical synthesis a protein of interest… !7 !8 Getting non-natural fluorophores into a cell Some sea creatures make natural fluorophores Trends Bioch. Sci., 1984, 9, 88-91. O O O N O N Bioluminescent - Fluorescent CO2 CO -- S 2 S C N NHN C H S S Chemically labeled proteins of interest Microinjection with micropipet O O O N O N Fluorescent - CO2 CO - S 2 N NH H S …and then manually injected into a cell Some natural fluorophores are genetically encoded proteins http://www.luminescentlabs.org/and can be transplanted into cells as DNA! 228 OSAMU SHIMOMURA, FRANK H.
    [Show full text]
  • Fluorophore Referenceguide
    Fluorophore Reference Guide Fluorophore Excitation and Emission Data Laser Lines Broad UV Excitation Excitation Maxima Emission Maxima Emission Filters 290-365 nm LP = Long pass filter DF = Band pass filter Excel. ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ DAPI: 359 nm ____ SP = Short pass filter Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ GFP (Green Fluorescent Protein): 395 nm ____ 400 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Coumarin: 402 nm ____ 425 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ AttoPhos: 440 nm ____ ____ 443 nm: Coumarin 450 nm Good ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Acridine Orange: 460/500 nm ____ ____ 461 nm: DAPI Good __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ R-phycoerythrin: 480/565 nm ____ Excel.
    [Show full text]
  • Water-Soluble Pyrrolopyrrole Cyanine (Ppcy) NIR Fluorophores† Cite This: Chem
    Erschienen in: Chemical Communications ; 2014, 50. - S. 4755-4758 ChemComm View Article Online COMMUNICATION View Journal | View Issue Water-soluble pyrrolopyrrole cyanine (PPCy) NIR fluorophores† Cite this: Chem. Commun., 2014, 50, 4755 Simon Wiktorowski, Christelle Rosazza, Martin J. Winterhalder, Ewald Daltrozzo Received 7th February 2014, and Andreas Zumbusch* Accepted 21st March 2014 DOI: 10.1039/c4cc01014k www.rsc.org/chemcomm Water-soluble derivatives of pyrrolopyrrole cyanines (PPCys) have been dyes, BODIPYs or others.7 Notable are also advances in other fields, synthesized by a post-synthetic modification route. In highly polar like the engineering of GFP-related fluorescing proteins or quantum media, these dyes are excellent NIR fluorophores. Labeling experiments dots, which have resulted in the synthesis of novel systems with NIR show how these novel dyes are internalized into mammalian cells. emission.8 To date, however, only a few water-soluble dyes with strong NIR absorptions and emissions have been known. Apart from the Near-infrared (NIR) light absorbing and emitting compounds have general scarcity of NIR absorbing molecules, the main reason for this attracted a lot of interest since the 1990’s.1 Initially, this was motivated is that NIR absorption is commonly observed in extended p-systems by their use in optical data storage or as laser dyes. Recently, however, which most often are hydrophobic. The incorporation of hydrophilic new applications of NIR dyes have emerged, which has led to a surge of functionalities into
    [Show full text]
  • Chemical Probes to Visualize Bacterial Cell Structure and Physiology
    molecules Review From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology Jonathan Hira 1, Md. Jalal Uddin 1 , Marius M. Haugland 2 and Christian S. Lentz 1,* 1 Research Group for Host-Microbe Interactions, Department of Medical Biology and Centre for New Antibacterial Strategies (CANS), UiT—The Arctic University of Norway, 9019 Tromsø, Norway; [email protected] (J.H.); [email protected] (M.J.U.) 2 Department of Chemistry and Centre for New Antibacterial Strategies (CANS), UiT—The Arctic University of Norway, 9019 Tromsø, Norway; [email protected] * Correspondence: [email protected] Academic Editor: Steven Verhelst Received: 30 September 2020; Accepted: 23 October 2020; Published: 26 October 2020 Abstract: Chemical probes have been instrumental in microbiology since its birth as a discipline in the 19th century when chemical dyes were used to visualize structural features of bacterial cells for the first time. In this review article we will illustrate the evolving design of chemical probes in modern chemical biology and their diverse applications in bacterial imaging and phenotypic analysis. We will introduce and discuss a variety of different probe types including fluorogenic substrates and activity-based probes that visualize metabolic and specific enzyme activities, metabolic labeling strategies to visualize structural features of bacterial cells, antibiotic-based probes as well as fluorescent conjugates to probe biomolecular uptake pathways. Keywords: activity-based probe; antibiotic conjugate; bacterial imaging; bacterial uptake; fluorogenic substrate; metabolic labeling; phenotypic heterogeneity 1. Introduction—From 19th Century Microbiology to Modern Day Chemical Biology If chemical biology can be defined as the ‘interrogation of biological systems with chemical approaches’ [1], we must acknowledge some of the first microbiologists as chemical biologists.
    [Show full text]
  • Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome
    Near-infrared genetically encoded positive calcium indicator based on GAF-FP bacterial phytochrome The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Subach,Oksana M., Natalia V. Barykina, Konstantin V. Anokhin, Kiryl D. Piatkevich, and Fedor V. Subach, "Near-infrared genetically encoded positive calcium indicator based on GAF-FP bacterial phytochrome." International Journal of Molecular Sciences 20, 14 (July 2019): no. 3488 doi 10.3390/ijms20143488 ©2019 Author(s) As Published 10.3390/ijms20143488 Publisher Multidisciplinary Digital Publishing Institute Version Final published version Citable link https://hdl.handle.net/1721.1/125333 Terms of Use Creative Commons Attribution Detailed Terms https://creativecommons.org/licenses/by/4.0/ International Journal of Molecular Sciences Article Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome 1, 2, 2,3 Oksana M. Subach y , Natalia V. Barykina y , Konstantin V. Anokhin , Kiryl D. Piatkevich 4 and Fedor V. Subach 1,* 1 National Research Center “Kurchatov Institute”, Moscow 123182, Russia 2 P.K. Anokhin Institute of Normal Physiology, Moscow 125315, Russia 3 Lomonosov Moscow State University, Moscow 119991, Russia 4 MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA * Correspondence: [email protected]; Tel.: +07-968-962-7083 These authors contributed equally to this work. y Received: 31 May 2019; Accepted: 15 July 2019; Published: 16 July 2019 Abstract: A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum.
    [Show full text]
  • Orientation of Cyanine Fluorophores Terminally Attached to DNA Via Long, Flexible Tethers
    1148 Biophysical Journal Volume 101 September 2011 1148–1154 Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible Tethers Jonathan Ouellet, Stephanie Schorr, Asif Iqbal, Timothy J. Wilson, and David M. J. Lilley* Cancer Research UK Nucleic Acid Structure Research Group, The University of Dundee, Dundee, United Kingdom ABSTRACT Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 50-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 50-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocya- nine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindo- carbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ~60 for the long- tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.
    [Show full text]
  • Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series
    bioRxiv preprint doi: https://doi.org/10.1101/2020.11.13.381152; this version posted November 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Gebhardt et al., Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series Christian Gebhardt1, Martin Lehmann2, Maria M. Reif3, Martin Zacharias3 & Thorben Cordes1,* 1 Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany 2 Plant Molecular Biology and Plant Metabolism, Faculty of Biology, Ludwig-Maximilians- Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany 3 Theoretical Biophysics (T38), Physics Department, Technical University of Munich, München, Germany corresponding author email: [email protected] Abstract The use of fluorescence techniques has had an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the availability of numerous commercial dyes, but often information about the chemical structures of dyes (and their linkers) are a well-kept secret. This can lead to problems for applications where a knowledge of the dye structure is necessary to predict (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of spectroscopy, mass spectrometry and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647).
    [Show full text]
  • Fluorescence Spectroscopy of Exogenous, Exogenously-Induced and Endogenous Fluorophores for the Photodetection and Photodynamic Therapy of Cancer
    FLUORESCENCE SPECTROSCOPY OF EXOGENOUS, EXOGENOUSLY-INDUCED AND ENDOGENOUS FLUOROPHORES FOR THE PHOTODETECTION AND PHOTODYNAMIC THERAPY OF CANCER. Thèse présentée au Département de Génie Rural Ecole Polytechnique Fédérale de Lausanne par Matthieu Zellweger Jury: Dr Georges Wagnières, rapporteur Prof. Hubert van den Bergh, corapporteur Dr Christian Depeursinge, corapporteur Prof. René Salathé, corapporteur Prof. Philippe Monnier, corapporteur Prof. Stanley Brown, corapporteur Lausanne, Février 2000 2 Foreword This work is the final report about my research activity as a Ph.D. student in the LPAS laboratory of Prof. Hubert van den Bergh, at the Swiss Federal Institute of Technology - Lausanne (EPFL). Some parts of this report have been published or submitted during the course of my Ph.D. thesis. I therefore included the text of the publications instead of an original chapter whenever relevant. In such a case, the reference is clearly stated. A brief introduction always precedes the resulting 'chapters'. Due to this inclusion, the reader might find some redundant information within this report. On the other hand, it should be emphasized that each chapter can be read as a stand-alone text and need not be linked to any other part of the whole work. However, I tried to avoid the unnecessary repetition of information and this is especially true for the references. Whenever the reader feels that a reference should have been included to support an information in the introduction, they should check the introduction of the following published chapter for a more detailed reference list. The same criticism applies to the introduction and, again, the reader should check the published section of a chapter if they feel that it lacks some information.
    [Show full text]
  • Reflectance and Fluorescence Spectroscopies in Photodynamic
    Reflectance and Fluorescence Spectroscopies in Photodynamic Therapy by Jarod C. Finlay Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Supervised by Professor Thomas H. Foster Department of Physics and Astronomy The College Arts and Sciences University of Rochester Rochester, New York 2003 To Leah iii Curriculum Vitae The author was born in Bryn Mawr, Pennsylvania, in 1975 and received his early education in the Pennsylvania towns of Phoenixville and Montoursville. During his junior year of high school, Finlay moved to Exeter, Pennsylvania where he enrolled in Exeter Township Senior High School. Here, he was first introduced to physics by his teacher Mr. Arthur Murray, whose encouragement of curiosity and inquisitiveness were more valuable than any lesson on Newton’s laws, and to higher mathematics by Mr. Robert Dahl, whose sense of humor made learning integral calculus an enjoyable experience. In his final year of high school, Finlay met Leah Janowsky, the woman he would later marry. After graduating high school in 1993, Finlay was accepted to Alfred University in Alfred, New York, with a National Merit Scholarship which allowed him the financial freedom to spend the next four years studying physics, mathematics, ce- ramic arts, and various humanities. His education in physics was supplemented by summer research projects at the Maria Mitchell Observatory and the University of Central Florida, both under the auspices of the National Science Foundation’s Research Experience for Undergraduates (REU) program, and by research con- ducted at Alfred under the tutelage of Professor David DeGraff. Finlay graduated CURRICULUM VITAE iv in the Spring of 1997 and was accepted to the graduate program in Physics at the University of Rochester with a Graduate Assistance in Areas of National Need (GAANN) Fellowship.
    [Show full text]
  • The Structure of the Chromophore Within Dsred, a Red Fluorescent Protein from Coral
    The structure of the chromophore within DsRed, a red fluorescent protein from coral Larry A. Gross*†, Geoffrey S. Baird‡, Ross C. Hoffman†§, Kim K. Baldridge§¶, and Roger Y. Tsien*†§ʈ *Department of Pharmacology, ‡Medical Scientist Training Program and Biomedical Sciences Program, †Howard Hughes Medical Institute, §Department of Chemistry and Biochemistry, and ¶San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA 92093 Contributed by Roger Y. Tsien, August 16, 2000 DsRed, a brilliantly red fluorescent protein, was recently cloned bonds. The resulting acylimine substituent quantitatively ac- from Discosoma coral by homology to the green fluorescent counts for the red shift by detailed quantum chemical calcula- protein (GFP) from the jellyfish Aequorea. A core question in the tions. It also explains why the DsRed chromophore shifts back biochemistry of DsRed is the mechanism by which the GFP-like to a GFP-like absorbance spectrum upon protein denaturation 475-nm excitation and 500-nm emission maxima of immature and why harsher treatments cleave DsRed into two fragments. DsRed are red-shifted to the 558-nm excitation and 583-nm emis- sion maxima of mature DsRed. After digestion of mature DsRed Materials and Methods with lysyl endopeptidase, high-resolution mass spectra of the Mass Spectral Analysis of LysC Digests. Approximately 2 nmol of purified chromophore-bearing peptide reveal that some of the His-tagged DsRed protein or its K83R mutant (1) was evaporated molecules have lost 2 Da relative to the peptide analogously to near dryness in vacuo then resuspended in 3 ␮lof6M prepared from a mutant, K83R, that stays green.
    [Show full text]