Bi9393 Analytická cytometrie Lekce 5 Oddělení cytokinetiky Biofyzikální ústav AVČR, v.v.i. Královopolská 135 612 65 Brno e-mail: ksoucek@ibp.cz tel.: 541 517 166 Karel Souček, Ph.D. K. Souček Bi9393 Analytická cytometrie Fluorescenční proteiny nbioluminescence resonance energy transfer (BRET) –Aequorea victoria - medúza žijící ve vodách na pobřeží Severní Ameriky. –je schopna modře světélkovat (bioluminescence). Ca2+ interaguje s fotoproteinem aequorinem. –modré světlo excituje green fluorescent protein. –Renilla reniformis – korál žijící ve vodách na severním pobřeží Floridy. –luminescence vzniká degradací coelenterazinu za katalytického působení luciferázy. –modré světlo excituje green fluorescent protein. – – Renilla reniformis "Sea Pansy" http://www.mbayaq.org/efc/living_species/default.asp?hOri=1&inhab=440 Aequorea victoria “Crystal jelly “ http://www.whitney.ufl.edu/species/seapansy.htm Fluorescenční proteiny http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm n n O2• – O2 H2O2 • OH H2O 4 e– reduction to water e– e– e– e– Not so terribly reactive with most biomolecules Maintained at very low concentration Catalases, peroxidases, GSH, etc… Actually a chemical reductant Not so terribly reactive with most biomolecules Mitochondrial superoxide the major source of active oxygen Maintained at very low concentration Superoxide dismutases Reacts with virtually any molecule at diffusion-limited rates The molecule that makes ionizing radiation toxic Unreactive at STP, but a great electron acceptor Biological activation via radicals, transition metals Generally, radical intermediates are enzyme-bound © Simon Melov DCFH-DA DCFH DCF COOH H Cl O O-C-CH3 O CH3-C-O Cl O COOH H Cl OH HO Cl O COOH H Cl O HO Cl O Fluorescent Hydrolysis Oxidation 2’,7’-dichlorofluorescin 2’,7’-dichlorofluorescin diacetate 2’,7’-dichlorofluorescein Cellular Esterases H2O2 DCFH-DA DCFH-DA DCFH DCF H O 2 2 Lymphocytes Monocytes Neutrophils log FITC Fluorescence .1 1000 100 10 1 0 20 40 60 PMA-stimulated PMN Control 80 © J. P. Robinson, Purdue University Fluorescent sensors for detection of H2O2 Variants & fusions npHyPer-cyto vector npHyPer-dMito vector –Duplicated mitochondrial targeting sequence (MTS) is fused to the HyPer N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. npHyPer-nuc vector –Three copies of the nuclear localization signal (NLS) fused to the HyPer C-terminus provide for efficient translocation of HyPer to the nuclei of mammalian cells [Fischer-Fantuzzi and Vesco, 1988] n n n „High Throughput Flow Cytometry“ nautomatizace + robotizace = urychlení a efektivita sběru dat (měření desítky vzorků za hodinu s minimálním zásahem operátora ) nvyužití principu vícebarevné analýzy K. Souček Bi9393 Analytická cytometrie Automatizované systémy měření vzorků Automatický karusel (autosampler) Adaptér pro nasávání vzorků z mikrotitrační desky logo_becton%20dickinson Automatizovaný „microsampler“ systém cytek K. Souček Bi9393 Analytická cytometrie Universal Loader - Lab Image https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcSG6MDDEyKW2lrEy-4pYcbhalq94g7mws3iCujkWC8szVo IZvRtvw The steps in a high-throughput fluorescence-microscopy experiment. Analysis [USEMAP] http://www.stanford.edu/group/nolan/ Garry Nolan Peter Krutzik „Fluorescent cell barcoding“ Fig 1 full size Krutzik PO, Nolan Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods. 2006 May;3(5):361-8. K. Souček Bi9393 Analytická cytometrie > Fig 3 full size Krutzik PO, Nolan Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods. 2006 May;3(5):361-8. K. Souček Bi9393 Analytická cytometrie Cytometric bead array (CBA) nMultiplexed Bead-Based Immunoassays nflow cytometry application that allows users to quantify multiple proteins simultaneously Multiplex microsphere‐based flow cytometric platforms for protein analysis and their application in clinical proteomics – from assays to results ELECTROPHORESIS Volume 30, Issue 23, pages 4008-4019, 3 DEC 2009 DOI: 10.1002/elps.200900211 http://onlinelibrary.wiley.com/doi/10.1002/elps.200900211/full#fig1 Microspheres can be immobilized with capture molecules and immunoassays can be performed. (A) Coupling reactions for microspheres: Microspheres with functional groups (carboxyl‐, thiol‐) can be used for immobilizing peptides, proteins, or amino group‐functionalized molecules; alternatively, streptavidin‐functionalized microspheres can be applied for biotin‐labeled capture molecules. (B) Standard capture sandwich assay: An individual assay involving an optically encoded microsphere immobilized with a specific capture molecule (antibody) is being carried out on the particle. After sample incubation, another analyte‐specific antibody (a so‐called detection antibody, which is biotinylated and usually recognizes epitopes that are different from that of the capture antibodies) is then applied to the reaction. The presence and quantity of the reporter tag (e.g. strepavidin‐phycoerythrin) allows the precise quantification of the reactions that occur. © This slide is made available for non-commercial use only. Please note that permission may be required for re-use of images in which the copyright is owned by a third party. CBA CBA nmultiplexing capabilities nspeed nincorporation of multiple assay formats nrapid assay development and reasonable cost nautomation Biologické aplikace průtokové cytometrie nCytogenetika –analýza chromozómů •karyotyp •sortrování –chromozómové DNA knihovny –FISH značení (chromosome painting) • K. Souček Bi9393 Analytická cytometrie Analýza a sortrování chromozómů Analýza a sortrování chromozómů nsynchronizace buněk – zisk metafázních chromozómů (colcemid, hydroxyurea) nizolace chromozómů nznačení DAPI nebo Hoechst vs. chromomycin A3 (CA3) nebo mithramycin n= celková DNA vs. G/C-bohaté oblasti n NCBI PubChem logo http://www.scienceclarified.com/Ca-Ch/Chromosome.html http://www.nccr-oncology.ch/scripts/page9243.html Analýza a sortrování chromozómů e-bang „Flow karyotype“ http://www.sanger.ac.uk/HGP/Cytogenetics/ ♂ ♀ Karcinom močového měchýře Sortrování chromozómů Pisum sativum Sortrování chromozómů Vicia faba Aplikace průtokové cytometrie v mikrobiologii nekologie npotravinářství nbioterorismus http://www.cyto.purdue.edu/flowcyt/research/micrflow/ Aplikace průtokové cytometrie v mikrobiologii Aplikace průtokové cytometrie v mikrobiologii nviabilita nmetabolické funkce nsortrování nanalýza aerosolů (Fluorescence Aerodynamic Particle Sizer (Flaps)) n Aplikace průtokové cytometrie v mikrobiologii nSortrování –EPICS + Autoclone® modul n Fluorescence Aerodynamic Particle Sizer (Flaps) http://www.dres.dnd.ca/ResearchTech/Products/CB_PRODUCTS/RD98001/index_e.html Průtoková cytometrie kvasinek nbuněčné dělení nviabilita nmembránový potenciál nrespirace nprodukce H2O2 ncitlivost k antibiotikům nseparace n Saccharomyces cerevisiae http://en.wikipedia.org/wiki/Image:Budding_yeast_Lifecycle.png http://www.sbs.utexas.edu/mycology/sza_images_SEM.htm Průtoková cytometrie kvasinek Průtoková cytometrie v hydrobiologii nstudium pico- a nano-fytoplanktonu (< 20 mM) nanalýza metabolických funkcí planktonu nstudium pigmentace (analýza chlorofylu a fykoeritrinu) Průtoková cytometrie v hydrobiologii Průtoková cytometrie v hydrobiologii nanalýza DNA http://planktonnet.awi.de/portal.php?pagetitle=assetfactsheet&asset_id=15127 http://www.soes.soton.ac.uk/staff/tt/ [USEMAP] Průtoková cytometrie v hydrobiologii http://www.cyto.purdue.edu/flowcyt/research/micrflow/sieracki/sierack2.htm Absorption Spectrum Emission Spectrum http://omlc.ogi.edu/spectra/PhotochemCAD/html/chlorophyll-a(MeOH).html Průtoková cytometrie bezobratlých nlze aplikovat běžné metodické přístupy a fluorescenční značky n nPříklady aplikací: –buněčný cyklus –cytotoxicita –apoptóza Long Tongue Tachinid Fly 240px-Miesmuscheln_Mytilus_2 Invertebrate Survival Journal [USEMAP] http://www.icms.qmul.ac.uk/flowcytometry/uses/insects/index.html nFigure 5. Representative flow-cytometry scatter plot of hemocytes from 25 oysters. Rebelo MdF, Figueiredo EdS, Mariante RM, Nóbrega A, et al. (2013) New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes. PLoS ONE 8(2): e57384. doi:10.1371/journal.pone.0057384 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057384 nFigure 6. Proposed model for hemocyte maturation, as seen by flow cytometry. Rebelo MdF, Figueiredo EdS, Mariante RM, Nóbrega A, et al. (2013) New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes. PLoS ONE 8(2): e57384. doi:10.1371/journal.pone.0057384 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057384 ex vivo flow cytometrie - limitace nOvlivnění některých vlastností buněk (morfologie, exprese znaků); nneumožnuje dlouhodobější studie buněčného metabolismu a buněčných interakcí (komunikace, adheze) v přirozeném tkáňovém mikroprostředí; ndalší: –nízká citlivost pro detekci vzácných buněčných subpopulací (1-10 buněk/ml ~ 5000 – 50000 buněk v 5 litrech krve dospělého člověka); –časově náročná příprava vzorku (hodiny, dny); –diskontinuita odebíraných vzorků. Cytometry part A 79A: 737-745, 2011 in vivo vizualizace - limitace nTloušťka tkáně Imaging Basics – Reporter Molecules •Biology Fluorescent dyes Cell Bacteria Cell2 Mouse No light Mouse Light Label Cells Label Bacteria Genetic Marker Quantum dots and Nanoparticles Fluorescent Proteins Label Proteins Luciferase Transfection Direct cell/protein labeling IVIS® Lumina XR – Hardware •Hardware •Customized for in-vivo imaging • •High sensitivity from 300-900 nm • •Large dynamic range Lumina XR_closed The IVIS® Lumina XR Imaging Chamber •Hardware • XR cutaway CCD Camera Cooled -90C Interchangeable Emission Filter Wheel Heated Sample Stage f/1 Lens & Aperture Light-tight and shielded imaging chamber Cesium Iodide (CsI(Tl)) scintillator Automated aluminum filter X-Ray Source (40kVp,100mA) Tungsten Anode logo ‹#› logo ‹#› The Value of X-Ray + Optical Imaging •Mechanics •The Question: Where is the source origin relative to the surface signal? • •The Problem: Tissue attenuation/ scattering makes 2D optical signals difficult to locate at a defined location. • •The Solution: A co-registered X-ray image provides a fixed anatomical reference, defining skeletal structure and soft tissue organs and enabling better localization of the optical signal. Bariumphoto.bmp BariumXR.bmp X-Ray Provides a Fixed Anatomical Reference logo ‹#› logo ‹#› Camera and Lens Settings are Analogous to Photography * Field of View (FOV) is dependent on the distance from the lens to the sample * Light collected is proportional to how long the shutter is open (exposure time) * Aperture (ƒ/stop) controls the amount of light collected * Digital pixel binning is possible on the CCD – alters sensitivity/resolution •Mechanics Aperture (f/stop) Shutter CCD Field of View Emission Filters logo ‹#› logo ‹#› Field of View •Mechanics FOV B = 7.5 x 7.5 cm FOV A = 5 x 5 cm FOV C = 10 x 10 cm FOV D = 12.5 x 12.5 cm logo ‹#› logo ‹#› Photons Diffuse Through Tissue – Surface Light Pattern is Recorded * Light traveling through tissue scatters many times creating a “fuzzy” image at the surface of the animal * The IVIS® views the diffuse image on the camera-facing (top) surface of the subject * Not all light from the source will make it to the camera – light absorption will occur as signal exits the animal •Mechanics CCD Optics Bioluminescent Source Mouse Line Art Mouse Line Art Mouse Line Art Christ with St. Joseph in the Carpenter's Shop Georges De La Tour, ~ 1640 (Musee du Louvre, Paris). File:La Tour.jpg http://osa.magnet.fsu.edu/teachersparents/articles/sourcesoflight.html logo ‹#› logo ‹#› Průchod světla tkání http://www.photobiology.info/PhotobioInArt_files/hand1.png The absorption spectrum of a human hand. The spectrum was recorded with a very sensitive spectrophotometer with the hand in close juxtaposition with the photocathode (unpublished data of Karl H. Norris, from The Science of Photobiology (KC Smith, ed., Plenum Press, 1977; p. 400). logo ‹#› logo ‹#› Effective penetration depth in breast tissue logo ‹#› logo ‹#› Challenge of in vivo Optical Imaging * Photons are absorbed and scattered in tissue * Surface signal depends on source depth * Tissue is both autoluminescent and autofluorescent * Autofluorescence levels are much higher that autoluminescence •Mechanics Autoluminescence Luminescent or Fluorescent source logo ‹#› logo ‹#› How an X-Ray Image is Acquired •Mechanics Mouse Line Art CCD Optics X-ray Source Scintillator Stage • X-rays will be attenuated in tissue differently resulting in an image on the scintillator • The CCD views the scintillator resulting in a planar X-ray image • X-ray and Optical images have different path lengths. To correct this geometrical difference, the X-ray image is registered to the optical image • • • Lumina XR scint open angle4_Crop.tif Close Up of Scintillator in Position logo ‹#› logo ‹#› Field of View (X-ray) •Mechanics FOV B 7.5 x 7.5 cm FOV A 5 x 5 cm FOV C 10 x 10 cm FOVB.bmp FOVA.bmp FOVC.bmp logo ‹#› logo ‹#› Optional Z-FOV (2.5) •Mechanics Macro_Front_Paw2.BMP ball socket.JPG Magnified Mouse Paw Magnified Hip Ball and Socket logo ‹#› logo ‹#› •Overview IVIS® Lumina Fluorescence Components IVIS Lunima Internal §Fully computer controlled § §User interchangeable eight position Emission filter wheel § §Twelve position Excitation filter wheel § §Low Auto Fluorescence optics and fiber optics § §150 Watt Tungsten/Halogen lamp with computer controlled intensity logo ‹#› logo ‹#› •Overview IVIS® Lumina XR C5VG7309 10 excitation filters IVIS Internal Rendering CCD Array Optical Filter Wheel Heated Sample Stage Electronics Tray Camera Cover f/1 Lens & Aperture Light-tight Enclosure Shutter logo ‹#› logo ‹#› In Vivo Fluorescent Agent Applications ProSense ProSense FAST CatB FAST MMPSense MMPSense FAST AngioSPARK AngioSense Neutrophil Elastase FAST RediJect 2-DG RediJect COX-2 Cat K FAST OsteoSense Arthritis IntegriSense Annexin Vivo Inflammation ReninSense GastroSense Oncology Bone Biology Hypertension Cardiovascular Gastric emptying * Agents support multiple in vivo research areas Infectious Disease Bacterial Detection Probe * tools for in vivo molecular imaging §Prostate, Breast, Melanoma, and Colon Carcinoma Models §for syngeneic immunocompetent mice strains C57Bl/6 or BALB/c §stable transfected with lentiviral luc vector §CT26 luc, GFP - mouse colon cancer in process §4T1 luc - mouse breast cancer done, tested in vivo §B16 F10 luc, GFP – mouse melanoma done, tested in vivo §TRAMP-C1 GFP, luc – mouse prostate cancer in process § §Future plan: transfect cells with pLKO.1-CMV-fLuc-IRES-mCherry § D:\Kaja\Desktop\various\4T1_B16 _ cut.jpg 4T1 luc - s.c. injection B16 F10 luc - s.c. injection C:\Users\ksoucek\AppData\Local\Temp\KS20121130100206-1.PNG GFP ex-vivo imaging: example logo huan health Color is a “pigment” of our imagination 74 Light has no color “Color” resides in the eye of the observer These two yellows are visually indistinguishable BUT multispectral imaging can be used to resolve their spectral differences Fluorescence unmixing in cell biological research Modern Research and Educational Topics in Microscopy. A. Méndez-Vilas and J. Díaz (Eds.) ©FORMATEX 2007 Modern Research and Educational Topics in Microscopy. A. Méndez-Vilas and J. Díaz (Eds.) ©FORMATEX 2007 Spectral unmixing: basic principle http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/images/spectralintrofigure8.jpg Modern Research and Educational Topics in Microscopy. A. Méndez-Vilas and J. Díaz (Eds.) ©FORMATEX 2007 Spectral unmixing: results IntegriSense 680 Fluorescent Imaging Agent IntegriSense™ 680 fluorescent imaging agent is used for in vivo detection of integrin avb3 using a low molecular weight peptidomimetic antagonist coupled to a red fluorochrome. IntegriSense 680, an integrin-targeted molecular imaging agent allows the non-invasive imaging of disease status and progression. Excitation unmixing: Setup Excitation unmixing: Setup Excitation unmixing: Raw data - Autoflurescence, GFP, AF680 Excitation unmixing: Autoflurescence, GFP, AF680 Excitation unmixing: Autoflurescence, GFP, AF680 D:\Kaja\Desktop\Bez názvu 1.jpg D:\Kaja\Desktop\Bez názvu 2.jpg Excitation unmixing: Autoflurescence, GFP, AF680 In vitro 3D imaging using Lumina XR: autofluorescence & GFP - B16 F10 GFP cells - seeded 100 000, 200 000, 300 000 cells per insert (bottom row, 3D) and the same per well (top row, 2D) - imaging using IVIS Lumina XR - excitation from the top, plate without the lid - data analysis using spectral unmixing http://www.reinnervate.com/assets/images/science.jpg Ex. 430/Em. 515-575 Ex. 465/Em. 515-575 In vitro 3D imaging using Lumina XR: autofluorescence & GFP 48 h 24 h 72 h IVIS Lumina XR souhrn Výhody: •možnost kombinace detekce luminescence, fluorescence, X-ray •intuitivní ovládání •option Z-FOV 2.5 x 2.5 cm •spektrální unmixing • Limitace: •omezený počet emisních filtrů •aktuální konfigurace FOV max. 12.5 x 12.5 cm (tendr option 24 x 23 cm) •zatím jen syngenní in vivo modely •X-ray pouze pro malé hlodavce a FOV max. 10 x 10 cm •3D object - > 2D image in vivo flow cytometry – základní principy nZobrazení buněk přímo v krevním nebo lymfatickém řečišti. nVizualizace pomocí CCD nebo CMOS kamery po ozáření konvenční mikroskopickou lampou nebo lasery. nDetekce absorbce, fluorescence, Ramanova spektra, fototermálních nebo fotoakustických signálů. n Cytometry part A 79A: 737-745, 2011 in vivo flow cytometry Cytometry part A 79A: 737-745, 2011 in vivo flow cytometry – bez značení nNahrávka videa pomocí vysokorychlostní CCD nebo CMOS kamery s vysokým rozlišením v režimu propustnosti nebo odrazu. nPříklad: high-speed transmittance digital microscopy (TDM) nLimity: hloubka tkáně. nTDM může sloužit k navedení zdrojů záření pro další analýzu do určené oblasti. n n Cytometry part A 79A: 737-745, 2011 photoacoustic and photothermal imaging nThe photoacoustic effect was first discovered by Alexander Graham Bell in his search for a means of wireless communication.1 Bell succeeded in transmitting sound with an invention he called the “photophone,” which carried a vocal signal with a beam of sunlight that was reflected by a vocally modulated mirror. The sound could be recovered with an ordinary telephone receiver connected to a selenium cell illuminated by the light. Bell published the results in a presentation to the American Association for the Advancement of Science in 1880. The Photoacoustic Effect Benjamin T. Spike Physics 325 April 21, 2006 Schematic illustration of photoacoustic imaging Cytometry part A 79A: 737-745, 2011 ACS Nano 2010 4 (8), 4559-4564 In vivo flow cytometrie – detekce specifických signálů nDetekce fotoakustických a fototermálních jevů Cytometry part A 79A: 737-745, 2011 in vivo flow cytometry - aplikace Shrnutí přednášky n„High-throughput“ průtoková cytometrie … n … a uplatnění vícebarevné detekce a beads array n sortrování chromozómů n aplikace v mikrobiologii, hydrobiologii a studiu bezobratlých nin vivo průtoková cytometrie nin vivo zabrazovací metody Na konci dnešní přednášky byste měli: 1. 1.vědět co je to „high-throughput„ průtoká cytometrie …a jak se v ní může uplatnit princip vícebarevného značení. 2.znát základní principy měření a sortrování chromozómů pomocí průtokového cytometru; 3.mít představu o možných aplikacích průtokové cytometrie v mikrobiologii, hydrobiologii a studiu bezobratlých; 4.rozumět limitům a principům in vivo zobrazovaní a in vivo průtokové cytometrie. K. Souček Bi9393 Analytická cytometrie