Advanced fluorescence microscopy techniques
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1 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP C-13/1/KONV Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017
2 Lecture Outline History of microscopes Fluorescence and Fluorochromes Fluorescence Microscope Confocal Microscope Applications of Confocal Microscope Ferhan Ayaydin
3 History of Microscopy "Emeralds are usually concave so that they may concentrate the visual rays. The Emperor Nero used to watch in an Emerald the gladiatorial combats." Pliny the Elder A.D
4 History of Microscopy The first known compound microscope, made by Zacharias and Hans Janssen in the 1590's. Public domain, Wikipedia Antoni van Leeuwenhoek was an amateur Dutch scientist who was granted for his discoveries in microscopy and high quality, but crude optical microscopes. Jeroen Rouwkema, Wikipedia
5 History of Microscopy The Robert Hooke s design was a functional improvement over the traditional motif, and even included a lighting apparatus to aid in specimen illumination Hooke's microscope, from an engraving in Micrographia. Public domain, Wikipedia Public domain, Wikipedia
6 History of Microscopy Edal Anton Lefterov, Wikipedia 18 th Century Microscopes
7 History of Microscopy Hannes Grobe, Wikipedia 19 th Century
8 History of Microscopy Public domain, Wikipedia Public domain, Wikipedia 20 th Century Microscopes
9 History of Microscopy Ferhan Ayaydin 21 st Century Microscopes
10 fluorescent minerals Hannes Grobe, Wikipedia
11 Fluorescence and Fluorophores white light TransControl, Wikipedia UV light Didier Descouens, Wikipedia the term fluorescence comes from the mineral fluorite
12 Jablonski diagram of absorbance, non-radiative decay, and fluorescence Fluorescence occurs when a molecule relaxes to its ground state following excitation Excitation: S0+hv S1 Emission: S1 S0+hv Stoke s shift Public domain, Wikipedia
13 Detection of proteins by Immunofluorescence Common Fluorochromes FITC Rhodamine Texas Red Cyanine dyes Novel AlexaFluor dyes wide spectrum, stable brighter and bleach resistant Maria Francia & Boris Striepen, University of Georgia, Wikipedia Jakodak, Wikipedia
14 Staining Organelles with Fluorochromes Nucleus DAPI Hoechst dyes Ethidium Bromide Propidium Iodide Acridine Orange Mitochondria Mitotracker Mitofluor dyes Nonyl acridine orange Golgi/ER ER tracker fluorescent Ceramide fluorescent Sphingomyosin Gerry Shaw, Wikipedia Lysozme Lysotracker
15 What if my research project requires a fluorescent dye that....specifically stains lung cancer cells? Public domain, Wikipedia
16 Novel fluorescent chemical discovery through combinatorial chemistry Ferhan Ayaydin
17 Discovery of novel live cell permeable fluorescent chemicals oil bodies mitochondria membrane mitochondria Ferhan Ayaydin and Soujanya Kuntam compounds > microarray scanner > confocal microscopy
18 Novel dyes to stain plant oil bodies in live cells B2 C6 Ferhan Ayaydin and Soujanya Kuntam
19 Green Fluorescent Protein :GFP GFP is a small protein (27 kd) and the DNA sequences coding for GFP can be manipulated by recombinant DNA technology to create gene fusion promoter GFP your favorite protein promoter your favorite protein GFP Sierra Blakely, Wikipedia
20 The Mechanism of Glow The GFP chromophore consists of a cyclic tripeptide derived from Ser-Tyr-Gly at positions in the protein and is only fluorescent when embedded within the fully folded, complete GFP molecule. EGFP: Ser65 to Thr mutation (UV to blue excitation) Nascent GFP is not fluorescent, since chromophore formation occurs posttranslationally. The chromophore is formed by a cyclization reaction and an oxidation step at Tyr66 that requires molecular oxygen Jim G, Wikipedia
21 Fluorescent Protein Color Variants YELLOW Fluorescent Protein (YFP) (Thr 203 to Tyr) Citrine variant is very bright relative to EYFP and has been demonstrated to be much more resistant to photobleaching, acidic ph, and other environmental effects Another derivative, named Venus, is the fastest maturing and one of the brightest yellow variant CYAN Fluorescent Protein (CFP) (Tyr66 to Tryptophan) BLUE Fluorescent Protein (BFP) (Tyr66 to His) RED Fluorescent Protein (RFP) Susano-o, Wikipedia
22 Artwork by Nathan Shaner, photography by Paul Steinbach, created in the lab of Roger Tsien in 2006, Wikipedia A San Diego beach scene drawn with an eight color palette of bacterial colonies expressing fluorescent proteins derived from GFP and the redfluorescent coral protein dsred. The colors include BFP, mtfp1, Emerald, Citrine, morange, mapple, mcherry and mgrape.
23 The biggest advantage of using GFP is... Ferhan Ayaydin
24 Fluorescence Microscope upright inverted Masur, Wikipedia Nuno Nogueira, Wikipedia
25 Fluorescence Microscope Henry Mühlpfordt, Krzysztof Blachnicki, Wikipedia
26 Mercury Arc Lamp UV IR DETECTOR filter block
27 Filter sets used in fluorescence microscopy emission filter filter cube excitation filter dichoric mirror Ferhan Ayaydin
28 Confocal laser scanning microscopy Danh, Wikipedia
29 Optical sectioning with confocal microscope Ferhan Ayaydin
30 Advanced Applications of Confocal Microscopy Protein Dynamics and Interaction A) Bleaching techniques FRAP, ifrap, FLIP B) Photoconversion techniques PA-GFP, Kaede/Kikume, PS-CFP, EosFP, DRONPA C) Protein-protein Interactions FRET, BiFC
31 A) Bleaching techniques: FRAP, ifrap, FLIP FRAP: Fluorescence Recovery After Photobleaching Selective Laser bleaching with Laser Scanning Confocal Microscope Fluorescence recovery Ferhan Ayaydin
32 A) Bleaching techniques FRAP, ifrap, FLIP FRAP: Protein Mobility Comparison protein X protein Y Ferhan Ayaydin
33 A) Bleaching techniques FRAP, ifrap, FLIP FRAP: Kinetics of Fluorescence Recovery immobile fraction half recovery: t1/2 mobile fractions
34 A) Bleaching techniques FRAP, ifrap, FLIP Diffusion-limited fluorescence recovery Reaction-limited recovery Public Domain, Wikipedia
35 A) Bleaching techniques FRAP, ifrap, FLIP ifrap: Inverse FRAP Region of interest (ROI) bleaching laser prebleach bleach recovery (outside of region of interest) Ferhan Ayaydin dissociation parameters of molecules can be measured
36 A) Bleaching techniques FRAP, ifrap, FLIP Ferhan Ayaydin
37 A) Bleaching techniques FRAP, ifrap, FLIP Ferhan Ayaydin
38 A) Bleaching techniques FRAP, ifrap, FLIP Ferhan Ayaydin
39 Ferhan Ayaydin
40 B) Photoconversion techniques PA-GFP, Kaede/Kikume, PS-CFP, EosFP, DRONPA Wikipedia
41 C) Protein-protein Interactions FRET, BiFC Ferhan Ayaydin
42 C) Protein-protein Interactions FRET, BiFC Ferhan Ayaydin
43 C) Protein-protein Interactions FRET, BiFC Ferhan Ayaydin
44 C) Protein-protein Interactions FRET, BiFC BiFC: Bimolecular Fluorescence Complementation Public domain,wikipedia
45 C) Protein-protein Interactions FRET, BiFC Discussion: What happens if we use half GFP, half CFP and half YFP? Multicolor Complementation Is that possible?
46 Public domain, Wikipedia
47 Thank you for your attention! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practiceoriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMOP C-13/1/KONV project.
Advanced fluorescence microscopy techniques
Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP-4.1.1.C-13/1/KONV-2014-0001 Advanced
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