Confocal Microscopy and Living Cell Studies Eva Bártová Institute of Biophysics Academy of Sciences of the Czech Republic Table 7.1 Different Types of Llight Microscopy: A Comparison Type of Microscopy Brightfield (unstained specimen). Passes Jight directly through specimen; unless cell is naturally pigmented or artificially stained> image has little contrast. Bright field (stained specimen). Staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved). Fluorescence. Shows the locations of specific molecules in the cell. Fluorescent substances absorb short-wavelength, ultraviolet radiation and emit longer-wavelength, visible light. The fluorescing molecules may occur naturally in the specimen but more often are made by tagging the molecules of interest with fluorescent molecules. Light Micrographs of Human Cheek Epithelial Cells ..JA- ' y. Type of Microscopy Phase-contrast. Enhances contrast in unstained cells by amplifying variations in density within specimen; especially useful for examining living, unpigmented cells. D ifferent ial- inte rferen ce-co nt rast (Nomarski). Like phase-contrast microscopy, it uses optical modifications to exaggerate differences in density ConfocaL Uses lasers and special optics for "optical sectioning." Only those regions within a narrow depth of focus are imaged. Regions above and below the selected plane of view appear black rather than blurry This microscope is typically used with fluorescently stained specimens, as in the example here. Copyijghl © Pearson Education, Inc., publishing as Benjamin Cummings https://inhabitatxom/5-bioluminescent-species-that-light-up-the-world/bioluminescent-fungus-2/ Introduction to Fluorescence Sir GeOrQe Gabriel StOkeS (1819 — 1903) http://rstl.royalsocietypublishing.org/content/142/463.fuH.pdf+html a British physicist and mathematician Lakowicz et al., 2006 Ishikawa-Ankerhold et al., 2012 Introduction to Fluorescence Perrin-Jablonski diagram (1935) https://vwwv.researchgate.net/Perrin-Jablonski-diagram-The-vibrational-manifold-associated-with-electron • ground state (singlet S0) • vibrational relaxation • internal conversion (IC) -> the lowest singlet state (S^ • intersystem crossing (ISC) -> triplet state (T^ Introduction to Fluorescence GFP-derived__mRFPT-derived EvolvedbySHM f \ /"--~^-X> Exc. 380 43*452 488 516 437,504 540 548 554 568 574 587 595 596 605 590 nm Em. 440 4T5Í505 509 529 537/562 553 562 581 585 596 610 620 625 636 648 nm mmtmim? m m m ro O O TI TI -T| TJ TJ TJ í i 3 10 B 3 3 C (O 3. Q. BD <0 3 3 O 3 O 3 S tu -i 3 3 3 3 3 g O Q o QJ (B OJ 2002 - 2006, 2003 - 2014 FiťSt Wit Qfteťrtl "microscope" Abbe equation EM Optical microscope for tlie by physicians during eye surgery Contocal principle Ccirfocal laser scanning jjlerojecg^ Multiphcton rrtierůíCůpy STEO/SIM Super-neiCiluTion mkruicopy SO-OCT PALM FPALM &TORIW Cherntitry ffobei Príie GFP o ^A Ctte m-fífjy Habet Prize -1 Fij*. ft Robert Hoote microscope (1665) £j 'J 1 Marvin L. Minsky (1927-2016) dl The Nobet Prize in Chemistry 2014 Eric Betzig, Stefan W. Hell, William E. Moerner Share this: The Nobel Prize in Chemistry 2014 m'1 Photo: A_ Mahmoud Eric Betzig Prize share: V3 Photo: A. Mahmoud Stefan w. Hell Prize share: 1/9 Photo: A. Mahmoud William E. Moerner Prize share: 1/3 The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Bet2ig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy". The microscope Interpupillary distance Eyepiece Revolving nosepiece Switch the objectives lObiective 4x, 10x, 40x, 100x Stage Put and hold specimen Condenser Coarse focus knob Fine focus knob Stage motion control knobs X-axis and Y-axis Fig : http://www.microscopvuxorn/rnuseum/labophot.html Resolving power of microscopes 6 COpyriflftt. 2012. UfliverSitv Of WflrfcatO. All RJQfltS Reserved. www.sciencelearn.org.nz Numerical Aperture (NA) • ability to gather light and resolve fine specimen detail at a fixed object distance http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html • most oil immersion objectives —> a maximum numerical aperture of 1.4 • the most common numerical apertures ranging from 1.0 to 1.35 Numerical Aperture (NA) The Abbe diffraction limit Airy Disk (a) iTube Lens Objective Point Source The Airy Disk and Point-Spread Function X-Z Intensity Distribution OPTICAL MICROSCOPY (Rayleigh criterion) http://www2.optics.rochester.edu/workgroups/novotny/snom.html Point-Spread Function http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html The Abbe diffraction limit https://phys.org/news/2016-09-quantum-mechanics-technique-rayleigh-curse.html http://www.kurzweilai.net/the-nobel-prize-in-chemistry-2014-beyond-the-diffraction-limit-in-microscopy Confocal Microscopy • basic concept of confocal microscopy (1950s) • advances in computer and laser technology Detector Pinhole Aperture- Fluorescence -Barrier Filter In-Focus Light Rays Dichromatic — Mirror —Photo multiplier Detector Out-of-Focus Light Rays Objective Laser Scanning Confocal Microscope| Optical Configuration Laser Excitation Source __t_ Light Source Pinhole Aperture ~I Focal —I-Planes Figure 2 Specimen* http://fluoview.magnet.fsu.edu/theory/confocalintro.html MK Marvin L. Minsky (1927-2016) 1. Laser Excitation Source 2. Reflected through dichroic mirror 3. Into lens (Objective) 4. Focussed to the point in specimen 5. Emitted light (from specimen) 6. Into same lens 7. Beam splitter 8. Detector (Photomultiplier) Confocal Microscope lojmir Peträh (1923) Confocal Microscope Scanning System Nipkow disk Galvanometer Scan Mirror Galvano meter Gal va no mete r | Figure Laser Beam Scan Mirrur Dual Galvanometer Sinqle Mirror Nipkow Disk Nipkow / Peträh Disk Architecture 30* Rotation ' •SO A Archimedean Spiral Figure 2 (a) Synchronized Image SIM (Structured Illumination Microscopy) 191^^9595959 D+4A 631 SIM (Structured Illumination Microscopy) Advantages • 2x increase in spatial resolution over wide-field microscopy -> lateral (in xy) -100 nm • 3D imaging at fast frame rate • labelling using conventional fluorophores • up to 3 simultaneous colour imaging (other super-resolution microscopy modalities are often limited to 2) Disadvantages • artefacts generated during image reconstruction • sensitive to out-of-focus light and so difficult on thick or too densely labelled samples. Stimulated emission depletion (STED) microscopy super-resolution microscopy overcomes the diffraction limit of light microscopy jfi% The Nobel Prize in Chemistry 2014 Eric Betzig, Stefan W. Hell, William E. Moerner Share this: The Nobel Prize in Chemistry 2014 Li Photo: A. Mahmoud Eric Betzig Prize share: 1/3 Photo: A. Mahmoud Stefan W. Hell Prize share: 1/3 Photo: A. Mahmoud William E. Moerner Prize share: 1/3 The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy". https://www.nobelprize.org/nobeljDrizes/chernistry/laureates/2014/ Stimulated emission depletion (STED) microscopy • switching off the fluorescence by intense laser light —► in outer regions of diffraction limited excitation focus • detected fluorescence in center excitation focus —► high resolution images Stimulated emission depletion (STED) microscopy Applications ❖ Structural analysis -> instead of Electron Microscopy (EM) ❖ Correlative methods -> combining AFM + STED ♦> Multicolor ♦> Live-cell (ONLY plasma membrane with organic dyes) —► RECENTLY: multicolor live-cell STED (pulsed far-red laser) http://www.spiegel.de/fotostrecke/sted-mikroskopie-scharfer-blick-in-die-nanowelt-fotostrecke-51431-13.html Single-Molecule Localization Microscopy (SMLM) Thorley et al., 2014 fBALM CLEM SMLM SIM T-REX RESOLFT STED STORM FPALM DyMIN STED dSTORM REDCue STED PALM SOFI Deconvolution Electron Microscopes Light Microscopes Maximum resolution is Useful magnification is up to 250,000x in TEM, 100,000x in SEM Wavelength is 1.0nm. Highly detailed images, and even 3D surface imaging. Can see organelles of cells, bacteria and even viruses. Maximum resolution is 200nm Useful magnification is around 1000x (1500x at best) Wavelength is between 400 700nm. See reasonable detail, with true colours. Good for small organisms, invertebrates and whole cells. sample e~ detector 111— i TEM 5,5 nm^ A V 1 I TEM SEM Electron beam passes through thin sample. Electron beam scans over surface of sample. Specially prepared thin samples are supported on TEM grids. Sample can be any thickness and is mounted on an aluminum stub. Specimen stage halfway down column. Specimen stage in the chamber at the bottom of the column. Image shown on fluorescent screen. Image shown on TV monitor. Image is a two dimensional projection of the sample. Image is of the surface of the sample SEM https://www.majordifferences.com/2016/08/difference-between-sem-an Laboratory of Cellular Biophysics (2009) Leica TCS SP-5 X Leica TCS SP-8 SMD Laser Scanning Confocal Microscope • cultivation chamber (5% C02 and • cultivation chamber (5% C02 and temperature control, temperature control, Live cell experiments) Live cell experiments) • WLL (470-670 nm, Image acquisition) • WLL (470-670 nm, Image acquisition, FLIM-FRET) • Argon laser (Fluorescence Recovery After • Argon laser (Fluorescence Recovery After Photobleaching, FRAP) Photobleaching, FRAP) • UV-lasers (355 nm and 405 nm, DNA repair • UV-laser (405 nm, FLIM-FRET) studies) • FLIM-FRET Department of Molecular Cytology and Cytometry Assoc. prof. Eva Bártová, Ph.D. Patient Tumor Surgery Biopsy Single Cell Suspension Low Passage Cell Line https://wwnw.biomol.eom/rockland-introduces-melanoma-cell-lines.h Eukaryotic cells (10 to 100 Mm) Cell Line Organism Origin Tissue Ha La Human Cervical Cancer 293-T Human Embryonic Kidney A-54S Human Lung carcinoma ALC Murine Bone Marrow CHO Hamster Ovary HB54 Hybridoma Hybridoma Microvilli Vacuolar Ribosi Cenkiolei I Mitochondria Smooth endoplasmic reticulum http://spectorlab.labsites.cshl.edu/nuclear-domains/, Pro. D L Spector Nucleolus Hete roch ro matin luorescence Recover After Photobleaching Fluorescence In Situ Fluorescence Lifetime Imaging (FLIM) rster Resonance Energ Transfer (FRET) Methods FISH: • to form a diagnosis, • to evaluate prognosis, • or to evaluate remission of a disease, such as cancer Examples of diseases: • chronic myelogenous leukemia, t(9;22)(q34;q11) • acute lymphoblastic leukemia, t(12;21) • Down syndrome • sperm cells: an abnormal somatic or meiotic karyotype • does not require living cells • quantified automatically (a computer counts) 2D and 3D FISH Down syndrome http://swissperinatalinstitute.com/en/4_genetisch.html Methods Transfection • transfer of non-viral genetic material into eucarytic cells Goal: to express a particular gene in the host cell Used: to study gene expression regulation, protein function, gene silencing or gene therapy Transient Transfection Tr.in-.rc;" r Lipid recent ■J pi ď š mid DMA [J Mix equal volume of Transffrtu'n and DMA solution rc JD-ito 20 minutes to farm DIM A-liposome complexes Mix; add DNA-lipotome conplexes; directly to cŕll*<100 |JI r24-well ůlatef I Aspirate medi The cells iuni from \ IIS \ Incubate overnight and test the expression http://www.biorad.com/webroot/web/images/lsr/ solutions/technologies/gene_expression/pcr/tec hnology_detail/gxt42_img1 .jpg Stable Transfection Wang et al., 2015 | Day 1: Culture cells Day 2: Transfect cells with plasmid Day 3: C hange c ul tu re médium with selection reagent (Zeocin or G41B) SO O 2 5o Day 4: Split cells with series dilution into new petri dishes Cells without ilable lUHMfoCtlOn will tK gridijíHIí c I ud it! in t-"is period Culture cells until single colony appears (The time is cell type dependent) Pick up single- cell colony inn ontt wan or s-wan plate Culture cells until confluence (The time is cell type dependent) Culture cells until confluence, and pick up pure cells for subCUitUrinCj and preservation (The time 1» cell type dependent) Transient Transfection Stable Transfection Photoconversion Dendra2: improved green to red photoswitchable fluorescent protein • derived from octocoral Dendronephthya sp. (Gurskaya et al., 2006) • low phototoxicity Photoconversion monitoring selective cell fate real-time tracking protein dynamics (movement, degradation, etc.) H4-Dendra2 ■ ■ Cvackova etal., 2009 Methods Fluorescence Recovery After Photobleaching (FRAP) Movement (exchange (un)bleached) of molecules • Diffusion • Active transport 0 10 20 30 40 Time of recovery (s) Stixovä et al., 2011 Fluorescence Recovery After Photobleaching (FRAP) (Im)mobile fraction td diffusion time Fj fluorescence before bleaching F0 fluorescence just after bleaching Foe fluorescence in bleached region after full recovery Mobility = diffusion coeff. D related to td diffusion time R = (F. - F0)/ (F, - F0) Reits and Neefjes, 2001 FRAP in UV-damaged chromatin with HP1p Heterochromatin protein 1 (HP1) • formation of transcriptionally inactive heterochromatin • three HP1 protein family members in humans HP1a, HPIpand HP1y, HPip in DSBs / 3T3 cells a to u c 9 o.s 5 _ 0.« 3 2 0.7 1 « m -i- heteroc hromati n/con Irol -•- heterochromatinJTSA 10 IS 20 Time (S) i. euchromatin/control +- euchromatinrrSA 10 « 20 m Time (s) > CD s ■- heterochromati decontrol Jl -•- euchmmatin/control 1S> u »> Time (s) heterochromatinrrSA euchromatin/TSA 15 20 21 Time (s) Sustackova et al., 2012 Methods Single particle tracking analysis Mean Square Displacement (MSD) Area of minimal enclosing ellipse (Lim2) HP1P HP1P-CONTROL HP1p-TSA HP10-Vorinostat ft * * # * > V 1 * HPtp-CONTROL HP1P-TSA 0.5 -0.5 -1 -0.5 0 0.5 1 HPip-Vorinostat 1 D.5 ....... % • 0 -D.5 ....... -1 -1 -05 0 0 5 [>im] HPlfj Peripheral foci: control 0.22 ± 0.12 (n = 10) Peripheral foci: TSA 0.13 + 0.10 (n = 19) Peripheral foci: actinomycin D 0.59 i : 0.4S (n = 4) Peripheral foci: vorinostat 0.17 i : 0.10 (n = 23) Central foci: control 0.15 ± 0.07 (n = 7) Central foci: TSA 0.16 ± 0.09 (n = 16) Central foci: actinomycin D 0.69 + 0.46 (n = 6) Central foci: vorinostat 0.21 ± 0.12 (n = 13) Area mean: control 0.19 d : 0.1 1 Area mean: TSA 0.14 i : 0.09 Area mean: actinomycin D 0.65 + 0.44 Area mean: vorinostat 0.18 i : 0.1 1 Stixova etal., 2011 Methods Immunofluorescence • fixed cells and tissues • specifically labeling biological macromolecules —► determine the localization and function of sub-cellular proteins, without affecting cell physiology The most common protocols: Direct Immunofluorescence YPrimary Antibody Second Antibody Indirect Immunofluorescence Example of staining of F-actin filaments (green) and nucleoli (red) in mouse fibroblasts (DNA blue) (G. Šustáčková) http://www.sinobiological.com/principle-of-immunofluorescence.html Nuclear envelopathies • a group of rare genetic disorders caused by mutations in genes encoding proteins of the nuclear lamina iRibosomes lamin B and lamin A/C/DNA J L Cytoplasm Huber and Gerace, 2007 SYNDROME SYMPTOMS MUTATION IN Atypical Werner syndrome Progeria with increased severity compared to normal Werner syndrome Lamin A/C Barraquer-Simons syndrome Lipodystrophy Lamin B Buschke-Ollendorf syndrome Skeletal dysplasia, skin lesions LEM domain containing protein 3 Cardiomyopathy dilated with quadriceps myopathy Cardiomyopathy Lamin A/C Charcot-Marie-Tooth disease Neuropathy Lamin A/C Emery-Dreifuss muscular dystrophy Skeletal and cardiac muscular dystrophy Emerin, Lamin A/C Hutchinson-Gilford progeria syndrome Progeria Lamin A/C Pelizaeus-Merzbacher disease Leukodystrophy Lamin B m c 1 . , 1 GFP-HP1ß/mCherry-!amin A I I I I J Broers et al., 2006 Sehnalova et al., 2014 5 min. Methods DNA repair studies ^^^^^^^^ DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. 1. an irreversible state of dormancy, known as senescence 2. cell suicide, also known as apoptosis (programmed cell death) 3. unregulated cell division, which can lead to the formation of a tumor that is cancerous Methods DNA repair studies Base- excision 1 1 Nucleotide excision 1 H Mismatch repair (BER) I^H 1 repair (NER) 1 Brepair (MMRi Single-strand damage Base Excision Repair (BER) • repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or deamination Nucleotide Excision Repair (NER) • recognizes bulky, helix-distorting lesions such as pyrimidine dimers and 6,4 photoproducts Mismatch Repair (MMR) • corrects errors of DNA replication and recombination that result in mispaired (but undamaged) nucleotides Hoeijmakers et al., 2001 Methods DNA repair studies Hoeijmakers et al., 2001 Double-strand breaks Non-Homologous End Joining (NHEJ) Homologous Recombination (HR) Microhomology-Mediated End Joining (MMEJ) Methods DNA repair studies Leica TCS SP-5 X Methods DNA repair studies • activation of DNA damage response (DDR) system • Phosphorylation Ser-139 residue histone variant H2AX (yH2AX) = early cellular response to induction DSBs Leica TCS SP-5 X UV-laser 355 nm UV-laser 405 nm 355 nm | BrdU sensitization without I BrdU sensitization I CPDs Q •** ** I ~"v , 53BP1 • 405 nm Hoechst sensitization without Hoechst sensitization Nucleotide excision repair Stixova et al., Folia Biologica, 2014 cyclobutane pyrimidine dimers (CPDs) GFP-H2B * —*. r i UVA-irradiation 0 min 5 h □ 6h T 20^ L_d 23h Legartova and Suchankova et al., JoVE, 2017 Methods DNA repair studies PCNA (Proliferating cell nuclear antigen) = a DNA clamp = processivity factor for DNA polymerase 5 = essential for replication • 53BP1 (Tumor protein p53 binding protein 1) = vital in promoting NHEJ pathway = protecting broken DNA ends from extensive resection A 0,5 min S min 15.5 mill 23 min 30.5 min Suchankova et al., 2015 Methods DNA repair studies 3 fnm i iT'rfn 4 x :n iff ryjuaras https://ibidi.com/gridded-dishes-slides/178--dish-35-mm-high-grid-500-glass-bottom.html Microirradiati ROIs ingle c Suchankova et al., 2015 < o en CO CO CD O CL ' \ ^ 1 . 1 v H 4-fl PI • ■ __ H □ / 4» -< Time interval nf protein eccurnjlatinrt E £0 I I ✓ r Living »11» ■ Protein accumulation ■ Maximum accumulation B Area of protein accumulation at DMA lesions Selected nrolglrj Methods Förster Resonance Energy Transfer (FRET) Ishikawa-Ankerhold et al., 2012 Methods Förster Resonance Energy Transfer (FRET) • a distance-dependent physical process by which energy is transferred nonradiativelv from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole-dipole coupling (Förster, 1965). http://www.molecular-beacons.org/toto/Marras_energy_transfer.html FRET Efficiency =--=-fi-6 * kFRET (DA)+k0ther(p) (l/r)6+ kother R°+r6 IA+ID http://research.chem.psu.edu/txlgroup/RESEARCH.html Methods Förster Resonance Energy Transfer (FRET) Fluorophore properties A good fluorophore • Large extinction coefficient (~ 105 cnr1M_1) • High fluorescence quantum yield ( > 0.8) • Large shift of the fluorescence vs. absorption (Stokes shift > 40 nm) • Low quantum yield of photobleaching ( < 10~6) https://imaqes.nature.com/full/nature-assets/nprot/iournal/v8/n2/ima .jpg Methods Förster Resonance Energy Transfer (FRET) Leica TCS SP5 X • protein-protein interactions FRET Acceptor Bleaching • donor "de-quenching" in presence of an acceptor • comparing donor fluorescence intensity in the same sample before and after destroying the acceptor by photobleaching FRETeff - (Dpost - Dpre)/ Dpost Legartova et al., 2014 Methods Förster Resonance Energy Transfer (FRET) Leica TCS SP5 X • protein-protein interactions FRET Acceptor Bleaching Sehnalova etal., 2014 Methods Förster Resonance Energy Transfer (FRET) Disadvantages of FRET • fluorescent probes + molecule of interest —► creation of fusion proteins = mutation and/or chemical modification of the molecules under study • speciment movement (during the bleaching procedure) • photo-bleaching once in sample • donor fluorophore emission bleed through —► acceptor emission channel Methods Fluorescence Lifetime Imaging (FLIM) - Förster Resonance Energy Transfer (FRET) Fluorescence Lifetime (x) • average time a fluorophore remains in excited state before returning to the ground state by emitting photon 1. Start the clock > laser pulse (picosecond frequency) 2. Stop the clock —► 1st photon that arrives at the detector 3. Reset the clock > wait for start next signal Absorption = 10"lss Dysli et al., 2017 S2 excited state S, excited state Fluorescence =10q si So ground state lager pulse III fluorescence, cnotwi a:B"-5tcp-i ve I 1 slart3lop-1lme 2 ' www.Dicoauant.com Fluorescence lifetime histogram Fit a exponencial decay -> get the fluorescence lifetime (in ns) E=l- * FRET t noFRET Methods Fluorescence Lifetime Imaging (FLIM) - Förster Resonance Energy Transfer (FRET) Time (ns) Legartova and Suchankova et al., JoVE, 2017 Methods Fluorescence Lifetime Imaging (FLIM) - Förster Resonance Energy Transfer (FRET) in i .Ji 11 i\\ i" i LI mil i. libit i _____j_ . TinD^-nioanT-w^TirííDr^írnnio-i-ní^nmiEi^-ín i-i-x-i-i-i-rg«iMCi™M4MM™iMmnr>«ňmM«n I TAD1 |TÄÖä^ PRD p mraiiíFiflsitiTsm™ äs ä 11 5 £ 5 S Relative mutation frequency in human cancer TET M REG I Functional domains Conserved domains Structure mutations - DNA binding ouiBi«i»B (Contact mutation) 53BP1 binding IMR90 R273C L194F D3 wt HDAC1dn WB:yH2AX Alexa 488 Alexa 594 yH2AX H3S10ph Overlay FRET Efficiency E [%] 5 Q CO Q a b 0 m * . * i fp • \ ' ■ '' * 5IIT 1 ) Phasor J?- Experiment \^ Experimental point Quenching trajectory / P2*^33\ I Pl\\ 1 1 A B Simple Rules for FRET: 1) If the experimental point lies on a straight line then it is NOT FRET 2) FRET efficiencies follow a "quenching trajectory" 3) Quantitative FRET efficiencies can be obtained from the position on the quenching trajectory https://biocenterat-my.sharepoint.eom/:p:/r/personal/lijuan_zhang_vbcf_ac_at/_layouts/15/Doc.aspx?sourcedoc=%7Bd0e7c7c8-a72e-42c6-8ff7-599b2235447f%7D&action=edit SCIENCE STUDENT How society sees me How religious people see me How it really ChiirLes íniv-erisilv in IVii^ue, 1st lu-cully »Y Mimikmi" Institute of Cellular Biology and Pathology _ miversity oi| če s ká mye lomová skupina Albcrttw Jr Piaha 2, CicdiEcpuUn: - 1CĽ--Ľ0 22i S6E flOI. e-mail Lgrr ^ LfL.cimi.u ^|£rOľlÍľl£CľL ^-t Department of Computer Graphics and Design E)(ípiiruneriti TJ3 J1014 KPGO Katedry Fsujlly ni Infcfmatits MU I The Research Council of Norway norway grants MINISTRY OF EDUCATION YOUTH AND SPORTS EUROPEAN COOPER AT [ON IN SCIENCE AND TECHNOLOGY ÜGACR CZECH 5CIÍNCE FOUNDATIOS mo: University of Oslo Marie Curie project PIRSES-GA-2010-269156 THE HEBREW UNIVERSITY OF JERUSALEM f*1 Department of Molecular Cytology and Cytometry Jana Krejčí, Gabriela Šustáčková, Andrea Horáková, Lenka Stixová, Petra Řezníčková-Podloučková, Jana Suchánková, Alena Svobodová, Denisa Komůrková, Michal Franěk, Jana Poláková, Veronika Janečková, Alžběta Kružicová, Jana Kůrová