Confocal Microscopy and Living Cell Studies Eva Bártová Institute of Biophysics Academy of Sciences of the Czech Republic History of microscopy: the light microscope (discovered by Robert Hook, 1665): an instrument that enables the human eye, by means of a lens or combinations of lenses, to observe enlarged images of tiny objects. It made visible the fascinating details of naked eye invisible worlds. 1957: Marvin Minsky patented the fist confocal microscope Types of light microscopes The bright field microscope is best known to students and is most likely to be found in a classroom. Better equipped classrooms and labs may have dark field and/or phase contrast optics. Differential interference contrast, Nomarski, Hoffman modulation contrast and variations produce considerable depth of resolution and a three dimensional effect. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications. Hoffmann’s modulation contrast Polarized Light Microscopy http://www.google.cz/imgres?imgurl=http:// www.microscopehelp.com/ Brno laboratory at IBP Tandem scanning microscopes based on Nipkow disk source of light sample CCD rotation pinholes in the disk dichroic mirror Confocal microscopy - principles AOTF and AOBS Scanning in 2D and 3D by confocal microscope Laser beam moves firstly along x axis and then starts with new line in y axis. y x y z Begin End x 2D 3D Finishing scanning of one thin optical slice in xy plane, the scanning plane is moved in z axis to other slice Optical resolution: conventional versus confocal Conventional Confocal Res = 0.61*λ / ΝΑλ / ΝΑλ / ΝΑλ / ΝΑ Res(xy) = 0.4*λ / ΝΑλ / ΝΑλ / ΝΑλ / ΝΑ Res(xz) = 0.45*λ /λ /λ /λ / n(1-cosα)α)α)α) Formulas by Kino 4PI and STED resolution are much higher… Pinaki Sarder and Arye Nehorai (2006) Deconvolution Assuming linearity, convolution of the object and the imaging system PSF is affected by noise and produces a blurred image. Deconvolution restores the original object to an improved resolution and higher signal-to-noise ratio (SNR) level. Types of fluorochromes Fluorochromes are essentially dyes, which accept light energy (e.g. from a laser) at a given wavelength and re-emit it at a longer wavelength. These two processes are called excitation and emission. 1. Fluorochromes conjugated with other molecule. Example represents quantum dots, used for for ultrasensitive nonisotopic detection. 2. Fluorochormes that binds directly to some structure. For example, DAPI or PI binds to DNA 3. Fluorochromes produced by organism like Aequorea victoria (GFP) or octocoral Dendronephthya sp. (Dendra2) Living Cell Studies - GFP technology Cheutin et al. Science (2003) Dendra2 photo-conversion Dendra2 is an improved version of a green-to-red photoswitchable fluorescent protein Dendra, derived from octocoral Dendronephthya sp. (Gurskaya et al., 2006). Leica TCS SP-5 X Incubation chambers for living cell studies Termistors in short tubes on the side of the chamber Partition with opening on the bottom of the chamber (for experiments under upright microscope) Cover glass 24x50 mm Glass tubes bent under the upper glass (to enable CO2 to flow above medium in the part II of the chamber) Part I – cells can be grown on both upper and lower sides of the chamber; this part is filled fully with medium Part II – medium reaches under the glass tubes; cells are not grown here Application of UV–laser (355 nm) in experimental studies on DNA repair DNA repair Single-strand damage Base excision repair (BER), which repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or deamination. Nucleotide excision repair (NER), which recognizes bulky, helix-distorting lesions such as pyrimidine dimers and 6,4 photoproducts. Mismatch repair (MMR), which corrects errors of DNA replication and recombination that result in mispaired (but undamaged) nucleotides. Double-strand breaks non-homologous end joining (NHEJ) microhomology-mediated end joining (MMEJ) homologous recombination (HR) Misteli and Soutogou (2009) DNA repair DNA repair foci Histone signature Brno nomenclature for histone modifications (Turner, 2005) Experiments of Gabriela Šustáčková, Eva Bártová and Lenka Stixová HP1ββββ / BMI1 Experiments of Gabriela Galiová Využití UV laseru 355 nm ke studiu DNA reparace Experiments of Gabriela Galiová Experiments of Gabriela Galiová and Lenka Stixová Šustáčková et al., JCP, 2011 ATP depletion Experiments of Gabriela Šustáčková DNA repair Chou et al., PNAS (2010) Experiments of Gabriela Šustáčková and Soňa Legartová FRAP in UV-damaged chromatin with accumulated BMI1 and HP1ββββ Experiments of Gabriela Šustáčková and Darya Orlova FRAP – BMI1 Experiments of Stanislav Kozubek Additional molecular-biology methods with application for confocal microscopy FRET (Fluorescence Resonance Energy Transfer) is a technique for measuring interactions between two proteins in vivo. In this technique, two different fluorescent molecules (fluorophores) are genetically fused the two proteins of interest. http://www.rsc.org/publishing/journals/ http://www.celanphy.science.ru.nl/ Bruce%20web/construction.htm EB group, IBP, Brno Bártová et al., JCS (2005) Bártová et al., JCS (2005) Bártová et al., JCS (2005) Single particle tracking Experiments of Lenka Stixová and Pavel Matula 3D-FISH a konfokální mikroskopie Maximální obraz Všech řezů Galerie optických řezů 3D reconstrukce CT Weierich et al., (2003) in press Image analysis and studies on nuclear radial distribution of selected genomic regions Comparative genome hybridization CGH onCGH on metaphasemetaphase spreadsspreads Comparative genome hybridization Advanced microscopic techniques Electron Microscopes are: scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the following information: Topography The surface features of an object or "how it looks", its texture; direct relation between these features and materials properties (hardness, reflectivity...etc.) Morphology The shape and size of the particles making up the object; direct relation between these structures and materials properties (ductility, strength, reactivity...etc.) Composition The elements and compounds that the object is composed of and the relative amounts of them; direct relationship between composition and materials properties (melting point, reactivity, hardness...etc.) Crystallographic Information How the atoms are arranged in the object; direct relation between these arrangements and materials properties (conductivity, electrical properties, strength, etc.) Transmission Electron Microscopes (TEM) Galiová et al. EJCB (2008) Scanning Electron Microscopes 4-pi microscopy (CC lab in Heidelberg) 4pi: improved axial resolution. The typical value of 500- 700 nm can be improved to 100-150 nm which corresponds to an almost spherical focal spot with 5-7 times less volume than that of standard confocal microscopy. beam spliter The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a beam splitter (BS) and directed by mirrors towards the two opposing objective lenses. At the common focal point superposition of both focused light beams occurs. Excited molecules at this position emit fluorescence light which is collected by both objective lenses, combined by the same beam splitter and deflected by a dichroic mirror (DM) onto a detector. There superposition of both emitted light pathways can take place again. Stimulated Emission Depletion microscopy, or STED microscopy, is a fluorescence microscopy technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction with standard confocal laser scanning microscopes and conventional far-field optical microscopes. STED SP-5 LSCM Leica STED – STimulated Emission Depletion SUPERRESOLUTION (subdiffraction) in xy plane Willig KI et al. Nature 2006 Kellner RR et al. Neurosience 2007 Sieber JJ et al. Biophy J 2006 Lin W et al. PNAS 2007 Kittel RJ et al. Science 2006 Seebach J Cardiovas. Res. 2007 Fitzner D et al. EMBO J 2006 Sieber JJ Science 2007 neurobiology membrane biology membrane rafts intracellular transport Hell, S. W. and J. Wichmann (1994). Opt. Lett. "Breaking the diffraction resolution limit by stimulated emission" Unique Super-resolution microscope systems from Carl Zeiss ELYRA combines PAL-M (Photo-activated localization microscopy) and SR-SIM technology Fluorescence microscopy technique comparison Objective Petri Dish Oil WFM CLSMSDCM SD6000: ~ 0.84 µm SP5: ~ 0.50 µm TIRF: 0.1 – 0.3 µmwidefield: ~ 1.13 µm TIRF moderately thick 5 µm – 30 µm thick > 30 µm ! does not matter ! Only evanescent waves thin samples < 5 µm moderately thick + deconvolution samples Z resolution measured 4PI: 0.1 – 0.12 µm! Confocal Laser Scanning Microscopy – advanced systems Leica TCS SP5 – universal system for everything! Leica TCS SP5 STED Leica TCS SP5-X WLL Leica TCS 4PI Leica DM6000 CFS – Confocal Fixed StageFRAP FRET AB, SE Live Data Mode ROI spectrophotometer APD SMD – FCS, FLIM, FCCS Spectral FLIM High Content Screening Auto 2-photon, 3-photon Unique Leica TCS SP5: the only broadband confocal Leica TCS SP5 basic features • full range of lasers: 355, 405, VIS, IR up to 1300 nm • conventional scanner up to 8192x8192 pxls • resonant scanner up to 29 f/s for 512x512pxls • AOBS – Acousto-Optical Beam Splitter • Up to 5 confocal spectral detectors • SuperZ Galvo and Pifoc Wide range of UNIQUE upgrades: • White Light Laser • Spectral FLIM • online ROI spectrometer • STED – super-resolution in xy plane Separate UNIQUE systems • Leica DM6000 B CFS - electrophysiology • Leica 4PI – high resolution in z axis Unique White Light Laser – set of lasers or just one tuneable source? No tunability Sub-optimal excitation Cross-excitation fixed Excitation Spectra 488nm 543nm 633nm (Alexa 488, Alexa 546, Alexa 568, Toto-3) Set of gas, DPSS or DL lasers Sophisticated merge module Expensive solution Only several combinations of wavelengths setting of excitation wavelength and intensity in software or at Panel Box „Smart Wavelength“ and „Smart Intensity“ Lambda Square Scan: lambda excitation and emission over whole visible spectra White Light Laser – new lambda scan, new wavelengths Concentration? Molecule motility? Molecule interaction? Diffusion behavior? Molecule identification? Reaction kinetics? Signal transduction? Explain phenomena! Predict models! Optimize agents! Improve procedures! Validate data! Understand life! Translocation? Verify assumptions! Quantify Life! – The Challenge Eva Bártová, Gabriela Šustáčková, Lenka Stixová, Soňa Legartová, Darya Orlova, Veronika Foltánková, Pavel Matula, Petra Sehnalová Institute of Biophysics, the Academy of Sciences of the Czech Republic, v.v.i., Brno Projects: Ministry of Education Youth and Sports of the Czech Republic; COST-CZ project LC11020. Grant Agency of the Czech Republic by grants Nos. P302/10/1022 and P302/12/G157. European Union project COST TD09/05 and EU-Marie Curie project PIRSES-GA-2010- 269156.