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. Brightheld (stained specimen). Staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved). Light Micrographs of Human Cheek Epithelial Cells 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. 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 ifferen t i ,> I - i ti ic rfcrth ce-co tit rasl (Nomarsbi). Like phase-contrast microscopy, it uses optical modifications to exaggerate differences in density. Confocal. Uses lasers and special optics for "optical sectioning.1' Only those regions within a narrow depth of focus are imaged. Regions above and below the selected plane of view appeal' black rather than blurry. This microscope is typically used with fluorescently stained specimens, as in the example here. Copvncjht© Pearson Education, Inc., pyMisnlngaseeniaminCummlngs https://inhabitatxom/5-bioluminescent-species-that-light-up-the-world/bioluminescent-fungus-2/ Introduction to Fluorescence [ 463 ] I XXX. On the Change of Uefrangibility of Light. By G. G. Stokes, M.A., F.U.S., Fellow of Pembroke. College, and Lucasiun Professor of Mathematics in the University of Cambridge. Sir George Gabriel Stokes (1819- 1903) a British physicist and mathematician Received May 11,—Read May 27, 1853 http://rstl.royalsocietypublishing.org/content/142/463.full.pdf+html blue-glass in church window excitation filter < 400nm Lakowicz et al., 2006 g g. stokes yellow-glass of wine emission filter transmits > 400nm Stokes shift absorption emission wavelength Ishikawa-Ankerhold et al., 2012 Introduction to Fluorescence Perrin-Jablonski diagram (1935) https://www research gate.net/Perrin-Jablonski the lowest singlet state (Sj) • intersystem crossing (ISC) -> triplet state (Tx) Introduction to Fluorescence Aequorea victoria GFP chromophoie 15 k G FP-derived mRFP1 -derived Evolved by SUM t--^ s~-- Exc. 380 «b*«e 468 516 540 548 554 568 574 587 595 596 605 590 nm Em1440 47asos509 529 5aT«*2 553 562 581 585 596 S10 620 625 636 648 nm If fill Iff I Uf TT TITl^^O M 7i 9. ?J it ZT £ SB F" O IB m 3 in 10 5 "O X5 =1 = =i S. m S- .3 return to ground state contain several combined aromatic groups, or plane or cyclic molecules with several tt groups not all energy is emitted as fluorescence, some is dissipated as heat or vibrational energy atv SO; Ishikawa-Ankerhold et al., 2012 Carl Zeiss Microjmaging GmbH http://www.atdbio.com/content/34/Alexa-dyes History of Microscopy: 2002 - 2006, 2008 - 2014 William E. Moerner The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William £. Moerner "for the development of super-resolved fluorescence microscopy". The microscope Interpupillary distance X-axis and Y-axis Fig : http://www.micrQscopyu.com/rnuseum/labophot.htnnl Resolving power of microscopes © Copyright. 2012. University of Watkoto, AH Rights Reserved, www.sciencelcarn.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 http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html 2n sin a 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 Co nfoca I Microscopy • basic concept of confocal microscopy (1950s) • advances in computer and laser technology Detector Pinhole Aperture— |F!uorescenee-FJarrier Fitter I it-Focus Light Rays —Photo multiplier Detector Laser Scanning Confocal Microscope| Optica J Out-of-Focus Configuration Light Rays Dichromatic Mirror Objective En citation Fitter Laser Excitation Source I Light Source Pinhole Aperture Figure 2 Specimen'' http://fluoview.magnet.fsu.edu/theory/confocalintro.html 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 J ^51 Mojmir Petrafi (1923) Confocal Microscope Scanning System Nipkow disk SIM (Structured Illumination Microscopy) 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 The Nobel Prize in Chemistry 2014 Eric Betzig, Stefan W. Hell, William E. Moerner Share this: The Nobel Prize in Chemistry 2014 kl n ^Hl 1 7« M fVmm A 'AM Photo: A. Mahmoud Eric Betzig Prize share: 1/3 Phono: A. Mafrmoud Stefan W. Hell Prize share: 1/3 Photo: A. Mahmoud William E. Moerner Pfize share: 1/3 The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner "far the development of super-resolved fluorescence microscopy". https://www.nobelprize.org/nobel_prizes/chemistry/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 f BALM CLEM SMLM SIM T-REX RESOLFT STED STORM FPALM DyMIN STED dSTORM REDCue STED PALM SOFI Maximum resolution is Maximum resolution is 200nm Useful magnification is up to 250,000xinTEM, 100,000x in SEM Useful magnification is around 1000x (1500x at best) Wavelength is 1.0nm. Wavelength is between 400-700nm. Highly detailed images, and even 3D surface imaging. See reasonable detail, with true colours. Can see organelles of cells, bacteria and even viruses. Good for small organisms, invertebrates and whole cells. TEM 5,5 nm4 i /V TEM SEM El e elm n beam passes lino ugh thin sample. EleeU'on 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 detector e sample SEM Leica TCS SP-5 X Leica TCS SP-8 SMD Laser Scanning Confocal Microscope cultivation chamber (5% C02 and temperature control, Live cell experiments) WLL (470-670 nm, Image acquisition) cultivation chamber (5% C02 and temperature control, Live cell experiments) 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 Patient Tumor t * LI áá Surgery J^ Biopsy I S i ngle Ce 11 S us pen si on Low Passage Ce 11 Li ne https://www.biomol.com/rockland-introduces-melanoma-cell-lines.ritml?id=1427 Eukaryotic cells (10 to 100 |jm) Cell Line Organism Origin Tissue HeLa Human Ceivical Cancer 293 T Human Embryonic Kidney A-549 Human Lung carcinoma ALC Murine Bone Marrow CHO Hamster Ovary HBE4 HybrkJoma Hybridoma http://spectorlab.labsites.cshl.edu/nuclear-domains/, Pro. D L Spector Methods FISH: NHGRI FACT SHEETS CAGCCGCAAGCGGAATTGGCGACATAA GTCGGCGTTCGCCTTAACCGCTGTATT CAGCCGCAAGCGGAATTGGCGACATAA GTCGGCGTTCGCCTTAACCGCTGTATT Denature and Hybridize Labeling with Fluorescent Dye GTRGCfiTGCHTGCHTGCRUIJ,! I I I I I I I I I I I I I I I GRTCGTRGGTCRGTCGTRl! I ' I I I I I I i I I I 1 I I I I I TflCGGfiGCGflGCGTflGCGTflGGTCRGGTCGRCGTRGCGTGI fiTCGRTCGTflGTCRTGCflTGCGflTGTRTTGflCGTflGGTCGGI GRGGCGRGGCGCGGRGTGCGTRGCGTRGGTCTGGTCGTRGI GTCRTGCGTRGCTRGCTRGCTRGGTCRGGCTRGTCRGTRTCi Probe DNA Probe DNA • 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 I Ax pi r.i to medium from The celili http ://www. bio rad .co m/we broot/we b/images/lsr/ solutions/technologies/gene_expression/pcr/tec hnology_detail/gxt42_img1 Jpg Stable Transfection | Day 1: cmtunecells- | Day 2: Transtett calls wlih p a&mid □ay 3: Change culture medium wJih seFactlon reagent [Zeocin or G41B) Wangetal., 2015 o i 5o Day 4: Split cells wfcth series dilution Into new petri dishes i.-. •- •• .• i. Stable :iI: en Will be In Ihrs period Culture cells until single colony ipf.esrs (The time is cell lype dependent) Pich up singl? cel mic onu wallers-1 Culture cells until confluence (The time is «11 type dependent) • • • • i - In eta well Into 6 aeIIe Culture cell* until confluence, and pick up pure cells for subculluring and preservation (The lime Is 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 e • 9 • e _ O Ä © 9 0 Cvackova et al., 2009 Methods Fluorescence Recovery After Photobleaching (FRAP) Movement (exchange (un)bleached) of molecules • Diffusion • Active transport Stixovä et al., 2011 Fluorescence Recovery After Photobleaching (FRAP) 1. (Im)mobile fraction 2. td diffusion time 3. Fj fluorescence before bleaching 4. F0 fluorescence just after bleaching 5. Fx fluorescence in bleached region after full recovery 6. Mobility = diffusion coeff. D —> related to td diffusion time FRAP in UV-damaged chromatin with HPip Heterochromatin protein 1 (HP1) • formation of transcriptionally inactive heterochromatin • three HP1 protein family members in humans HP1a, HP1(3and HP1y, HP1(5 in DSBs/3T3 cells a> 10 o e 1 " « 01 0.4 . 06 rr 0.4 -♦- heterochromatin/TSA euchromatin/TSA o s 10 15 20 li 30 Time (s) Sustackova et al., 2012 Methods Single particle tracking analysis Mean Square Displacement (MSD) Area of minimal enclosing ellipse (um2) HP1P-C0NTR0L HP1P-TSA HPip-Vorinostat 0 5 -0.5 -1 HPIp-CONTROL -1 -0.5 0 0.5 1 [urn] -1 HP1[1-TSA -1 -0.5 0 0.5 1 [jim] HP1P -Vorinostat -0.5 0 0.5 HPIp Peripheral foci: control 0.22 ± 0.12 (n = 10) Peripheral foci: T5A 0.13 ± 0.10 (n = 19) Peripheral foci: actinomycin D 0.59 ± 0.48 (n = 4) Peripheral foci: vorinostat 0.17 ± 0.10 (n = 23) Central foci: control .'■ 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 = i ■ Area mean: control 0.19 ± 0.11 Area mean: TSA 0.14 ± 0.09 Area mean: actinomycin D 0.65 ± 0.44 Area mean: vorinostat 0.18 ± 0.11 Stixova et al., 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 Primary Antibody Second Antibody Indirect Immunofluorescence ff 0 Primary Antibody Second Antibody Example of staining of F-actin filaments (green) and nucleoli (red) in mouse fibroblasts (DNA blue) (G. Šustáčková) Tissue or cell 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 lamin B and lamin A/C/DNA 1 1 r v1 m 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 GFP-HP1[l/mCherry-lamin A Broers et al., 2006 Sehnalova et al., 2014 vj i i • # -* /f7) ROI » » 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) ■ repair (NER) ■ Brepair (MMR) 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 dinners and 6,4 photo products 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 Double-strand breaks Non-Homologous End Joining (NHEJ) Homologous Recombination (HR) Microhomology-Mediated End Joining (MMEJ) Direct reversal repair (DRR) Homologous recombination (HR) Nonhomologous end joining (NHEJ) Single-strand annealing (SSA) Transiesion synthesis (TLS) Break-induced replication (BIR) Hoeijmakers et al., 2001 Methods DNA repair studies Irradiation experiment 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 Nucleotide excision repair Stixova et al., Folia Biologica, 2014 cyclobutane pyrimidine dimers (CPDs) Legartova and Suchankova et al., JoVE, 2017 Methods DNA repair studies PCNA (Proliferating cell nuclear antigen) = a DNA clamp = processivity factor for DNA polymerase o" = essential for replication 53BP1 (Tumor protein p53 binding protein 1) = vital in promoting NHEJ pathway = protecting broken DNA ends from extensive resection Suchankova et al., 2015 Methods DNA repair studies https://ibidi.com/gridded-dishes-slides/178--dish-35-mm-high-grid-500-glass-bottom.html Suchankova et al., 2015 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 nonradiatively from an excited molecular fluorophore (the donor) to another 1 i by means of intermolecular long-range dipole-dipole coupling (Förster, 1965). http://www.molecular-beacons.org/toto/Marras_energy_transfer.html FRET Efficiency = -^™-= , , ^ = -ß- * kFRET (DA)+k0ther(D) (l/l")6+ kother RJ+r6 IA+1D http://research.chem.psu.edu/txlgroup/RESEARCH.html Methods Förster Resonance Energy Transfer (FRET) Fluorophore properties A good fluorophore • Large extinction coefficient (~ 10s cnrr1M-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) |d Spectral overlap No FRET Donor Acceptor emission excitation Donor Acceptor emission excitation Correct orientation No FRET 405 nm Distance <10 nm No FRET 405 nm nm 4// nm \ 405 nm FRET ■ I < o CD m TJ C creation of fusion proteins = mutation and/or chemical modification of the molecules understudy • speciment movement (during the bleaching procedure) • photo-bleaching once in sample • donor fluorophore emission bleed through —> acceptor emission channel Methods j (FLIM) - r (FRET) Fluorescence Lifetime (t) • 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 Dysli etal., 2017 • Fluorescence lifetime histogram • Fit a exponencial decay -> get the fluorescence lifetime (in ns) £=\_ T FRET InoFRET 3^54 Methods j (FLIM) - r (FRET) Methods j (FLIM) - r (FRET) Disadvantages of FLIM • high repetition rate vs. long decay —> fluorescence decay in pulse period • count rates - pile-up problem —> „dead time" of electronics 0.1 I-1-1-1-1-1-■-■-■-1-■- 0 20 40 60 SO 1W 120 | www.picoquant.com SOLUTION: keep probability of detecting more than one photon per laser pulse low Methods Enrico Gratton Professor of Biomedical Engineering and Physics Laboratory for Fluorescence Dynamics University of California, Irvine — If The challenges of FLIM • At evc.y pixel there are contributions of several fluorescent species, each one could be multi-exponential. • To make things worse, we can only collect light for a limited amount of time (100-200 microseconds per pixel) which result in about 500-1000 photons per pixel. • This is barely enough to distinguish a double exponential from a single exponential decay. • Resolving the decay at each pixel in multiple components involves fitting to a function, and is traditionally a complex computational task "for experts only". A major problem is data analysis and interpretation Phasor Universal circle Phasor Experimen If at \ ■'" •.... -N / k ll in m J g 1 1 g= M*cos((|)) Experimental point * Quenching trajectory 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 my friends see me How my family sees me Keep gods Out of j Government! How I see myself How society sees me How religious people see me How it really is )lecular Cytology and Cytometry EUROPEAN COOPERATION IN SCIENCE AND T EC H NOLO1 mGA Marie Curie project PIRSES-GA-2010-269156 Assoc. prof. Eva Bártová, Ph.D. 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