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College of Medicine, Biological Sciences and Psychology Core Biotechnology Services (CBS) Advanced microscopy and bio-imaging I Dr Kees Straatman Manager of Advanced Imaging Facilities (AIF) 10 -12 am: Introduction Fluorescence microscopy Advanced imaging systems 2 – 4 pm : Fluorescence imaging Advanced fluorescence techniques Facility April 2013 Renaissance in biological imaging Genomics, proteomics, metabolomics Anti-body development Introduction of more stable fluorochromes Introduction of fluorescent proteins Introduction of more sensitive/faster detectors Introduction of new imaging platforms Advances in computer development/data processing April 2013 Renaissance in biological microscopy Whole animal imaging systems (Maestro, CRi) R. Weissleder, U. Mahmood and J. Tam, Massachusetts General Hospital Super resolution microscopy; STORM images of mammalian mitochondria. Zhuang Research Group, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA April 2013 Renaissance in biological imaging Combination of existing and new technologies IVIS Spectrum Bioluminescence and Quantum MicroCT scanners April 2013 Renaissance in biological imaging Combination of existing and new technologies Correlative Light and Electron Microscopy (CLEM) Daguenet et al. (2012) Mol Biol Cell 23: 1765-1782 April 2013 Simple microscope Hans Loncke Two Trinacria species of about 0,1 mm size Van Leeuwenhoek’s microscope Around 1668 July 2007 Micscape Magazine April 2013 Compound microscope Microscope built by Zacharias Janssen, probably with the help of his father, in the year 1595. Considered the first microscope. Robert Hooke. First to use the word ‘cell’ while looking at a piece of cork. First publication with drawing of a microorganism (microfungus Mucor) April 2013 Compound microscope CO2 and temperature control Incubator Joystick to move stage Light source (laser) Camera Antivibration table April 2013 Aberrations Around 1668 Up to 275x mag 1595 3-9x magnification April 2013 Aberrations Objective lens Chromatic correction Plan Achromat Blue (486 nm) and red (656 nm) Plan Fluorite blue, red and green (588 nm) Plan Apochromats Also corrected for 436 nm SuperApochromats (SAPO; Olympus) CFI Plan Apochromat VC - Nikon Infinity Colour-Corrected - Zeiss U-V-I – Leica Corrected from UV to the near infrared region Plan: flat-field /spherical aberration corrections April 2013 Aberrations April 2013 Aberrations Coverslip correction April 2013 Aberrations Coverslip correction Coverslip number Coverslip thickness (mm) #0 0.08 - 0.12 #1 0.13 - 0.17 #1.5 0.16 - 0.19 #2 0.17 - 0.25 #3 0.25 – 0.35 #4 0.43 – 0.64 Calculated intensities using a dry objective April 2013 Image Quality The quality of a microscope image is assessed by the following: Focus - Is the image blurry or well-defined? Resolution Spatial resolution: the ability to visualize two points as separate points Temporal resolution: frequency at which images are recorded/captured April 2013 Resolution 1872: Ernst Abbe formulates his ‘wave theory of microscopic imaging’ : d = λ 2η sin η = refractive index of medium η.sin = NA (numerical aperture) diffraction limited microscope April 2013 Resolution 1872: Ernst Abbe formulates his ‘wave theory of microscopic imaging’ : d = λ 2η sin η = refractive index of medium η.sin = NA (numerical aperture) d = 0.5 * λ NA Refractive index (η ): the light-bending ability of a medium. April 2013 Resolution April 2013 Resolution We want to resolve 2 points! The best focused spot of light that a perfect lens with a circular aperture can make, is limited by the diffraction of light. Diffraction limited microscope R = 0.61λ /NA (Rayleigh criterion) d = 0.5 λ/NA (Abbe) http://micro.magnet.fsu.edu/primer/java/imageformation/rayleighdisks/index.html April 2013 Resolution • Radial resolution: d = 0.5λ /NA R = 0.61λ /NA 400 nm 488 nm 633 nm 300 250 200 633 150 488 100 400 50 0 15 36 57 78 99 120 141 162 183 204 225 246 267 288 309 330 Excitation: April 2013 Resolution April 2013 Resolution Axial resolution: XY YZ d = 2λη/(NA)2 λ XY (nm) Z (nm) 488 231 754 561 244 867 XY (2D): pixel XYZ (2D): voxel (NA = 1.4; η = 1.515) XZ April 2013 Objectives d = 0.5 λ /NA R = 0.61 λ /NA But magnification has influence on optimal pixel size of CCD camera. Size of field of view for a fixed CCD chip. April 2013 Köhler illumination To obtain optimum contrast and resolution in brightfield microscope •Focus your sample •Close the field diaphragm •Focus the condenser Adapted from: http://biology.fullerton.edu/facilities/em/BrightSetup.html April 2013 Köhler illumination To obtain optimum contrast and resolution in brightfield microscope •Focus your sample •Close the field diaphragm •Focus the condenser •Centre the condenser •Open field diaphragm till whole view is filled Adapted from: http://biology.fullerton.edu/facilities/em/BrightSetup.html April 2013 Contrast No colour and too little contrast between structures with similar transparency. Solution 1: phase contrast microscope: first described in 1934 by Dutch physicist Frits Zernike; Nobel Prize for Physics, 1953 . It translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. April 2013 Phase contrast April 2013 Contrast No colour and too little contrast between structures with similar transparency. Solution 2: Differential interference contrast (DIC) microscopy uses polarized light with specialized beamsplitting (modified Wollaston or Nomarski) prisms. April 2013 DIC Only single cell or thin layer of cells are observable. Two other options: Darkfield microscopy Polarization microscopy April 2013 Contrast No colour and too little contrast between structures with similar transparency. Solution 3: stain your sample with colour dyes. Solution 4: Fluorescence microscopy Blue = DNA; Green = talin; Red = actin; Hematoxylin stain (histology) April 2013 Objectives April 2013 Objective colour codes April 2013 Fluorescence microscopy Advantages – – – – Very sensitive (can detect single molecules) Can be used in vivo Localization of proteins Good time resolution Disadvantages Usually requires a fluorescent label Excitation light can be damaging (phototoxicity, bleaching) Often time consuming Quantitative imaging is challenging April 2013 Luminescence Excitation of a molecule resulting in emission of light. Chemoluminescence: resulting of a chemical reaction Bioluminescence: by a living organism April 2013 Bioluminescence Best know is the firefly luciferase : Firefly Luciferase (reporter gene) luciferin + ATP + O2 Oxyluciferin + AMP + PPi + CO2 + light Emission peak ~ 560 nm (PPi = pyrophosphate) Renilla luciferase Coelenterazine + O2 Coelenteramide + CO2 + light Emission peak ~ 480 nm April 2013 Bioluminescence The green-emitting luciferase was derived from a Japanese luminous beetle (λmax 560 nm); the redemitting luciferase was derived from railroad worm (λmax = 630 nm) Kwon et al. (2010) BioTechniques 48: 460-462 April 2013 Luminescence Excitation of a molecule resulting in emission of light. Chemoluminescence: resulting of a chemical reaction Bioluminescence: by a living organism Photoluminescence: absorption of photons causing re-radiation of photons Phosphorescence: delayed radiation Fluorescence: instant radiation April 2013 Fluorescence Jablonski diagram Absorption of light with a short λ resulting in emission of light with a longer λ (The so-called Stokes shift) Fluorochrome: molecule that is fluorescent. Fluorophore: a component of a molecule which causes this molecule to be fluorescent. April 2013 Fluorescence microscopes April 2013 Filters The Stokes shift we can use to separate the excitation and emission light in fluorescence microscope 41 Filters • Exciter D470/40x • Dichroic 495DCLP • Emitter E515LPv2 Old filter April 2013 Filters • Exciter ET470/40x • Dichroic T495LP • Emitter ET525/50m New filter April 2013 Filters • Exciter s D350/50x; • Dichroic 62002BS • Emitter 61002m • S492/18x; • S572/23x April 2013 Filters Filter wheel April 2013 Filters Prism based Borlinghaus and Kuschel; Nature Methods - 3, (2006) diffraction grating April 2013 Light sources Arc bulb Mercury Arc lamps (e.g. X-cite) Xenon Arc lamps Metal Halide Arc lamps LED (light-emitting diode ) Monochromator Laser April 2013 Light sources Arc bulb Need long time to warm up Flickering, increases over time. Associated with inadequate cooling of the lamp. Most unstable light source in use for microscopy these days April 2013 Light sources Arc bulb Xenon is relatively weak but has a more continuous and uniform spectrum what makes it more preferable for quantitative imaging April 2013 Light sources LED • No warming-up/cooling-down • Fast switching • Low power consumption • High emission stability (does not change with brightness) • Extremely long life span • Minimal heat output • Compact size April 2013 Light sources Monochromator April 2013 Light sources Laser • Single wavelength • High power • White laser (supercontinuum laser) • A laser can be focused or non-focussed April 2013 Detectors CCD (charge-coupled device) EM-CCD (electron-multiplying CCD) CMOS (complementary-metal-oxide-semiconductor detector); much higher readout speed. PMT (photomultiplier tube) April 2013 Detectors 3 types of noise systems: Dark current noise – noise from heat and cosmic noise exposure dependent. Reduce by cooling camera. Read noise – noise of reading the signal - fixed; not PMTs Photon shot noise – square route of signal - signal dependent Signal to noise (S/N or SNR) is a measure for the quality of the system All values must be compared in electrons SNR = QE*S / V(NoiseDark) 2 + (NoiseRead)2 + (QE*S) S = Signal in Photons QE = Quantum efficiency Online Signal to Noise calculators exist; see e.g. www.photomet.com April 2013 Detectors • PMT (image pixel by pixel) A small fraction of the collected photons (less than 30%) generate photoelectrons which get amplified by a factor of about 1 million; Depending on the voltage. Dark noise; single scan with standardized gain and offset without sample Photon shot noise; square root of signal SNR = S/V(NoiseDark)2 + (NoiseShot)2 Alternative: SNR = (Signal – Background)/Standard Deviation of Background April 2013 Detectors CCD read out CMOS reads out pixel by pixel Fast Noise/pixel EM-CCD camera has an Amplification stap before output node More sensitive Extra noise (excess noise factor) Output node Read noise (fixed) April 2013 Detectors Cooled to -70 to -90 °C Reduced dark current Coates et al (2009) Scientific CMOS Technology A High-Performance Imaging Breakthrough White Paper April 2013 Detectors Photometrics Noise limited image i.e. the signals below the noise cannot be seen EMCCD: minimizing the read noise and dark current April 2013 Detectors EM-CCDs Hamamatsu April 2013 CCD Spatial Resolution Image Pixels Digital Image To meet resolution of the microscope, pixel size should be at least 1/2 airy disk size; ideally 1/3. Known as The Nyquist criterion: the minimal sampling density needed to capture ALL information from the microscope into the image. However, smaller pixels collect less photons, have less full well capacity and results in slower read outs of the chip. April 2013 Optimal resolution CCD Objective NA Projected size on CCD (µm2) (R * Mag.) Optimal pixel size (µm2) 40x 1.3 10.4 3.5 – 5.2 60x 1.4 14.4 4.8 – 7.2 100x 1.4 24.0 8 - 12 R = 0.61λ /NA with λ = 550nm Camera pixel size = 6.45x6.45 µm. So without much loss of resolution you could image with a 2x2 binning using the 100x objective April 2013 Binning Binning increases image intensity while decreasing resolution & transfer time April 2013 Binning 50ms, no binning Intensity max 746 Resolution 240nm 50ms, 2x2 bin Intensity max 2297 Resolution 480 nm Improved S/N Images collected by JWS in the Nikon Imaging Center at Harvard Medical School April 2013 Image Quality The quality of a microscope image is assessed by the following: Noise – S/N Focus - Is the image blurry or well-defined? Resolution – What is the minimal distance between two points in the image that still can be seen as two separate points? Brightness - How light or dark is the image? Contrast - What is the difference in colour and light between adjacent areas of the specimen? April 2013 Bit Depth vs. Dynamic Range Bit Depth is determined by how many electrons are used to equal one gray level. As bit depth increases, the same original information is divided into ever smaller increments. Bit depth Greyscale levels 1 2 2 4 4 16 8 256 12 4,096 16 65,384 April 2013 Exposure Time 687 1051 1858 3260 3888 Image maximum grayscale value 12-bit camera maximum = 4095 Increasing exposure time increases signal Images collected by JWS in the Nikon Imaging Center at Harvard Medical School April 2013 Gain (CCD) Increasing gain reduces the number of photons / gray scale value Increasing gain, same exposure time Images collected by JWS in the Nikon Imaging Center at Harvard Medical School April 2013 Advanced imaging systems Haze and blur • In a normal epi-fluorescence microscope you collect light from the focal plane and light from below and above the focal plane. Epi-fluorescence Perfect focus April 2013 Epi-fluorescence microscope Live cell imaging Multiple colours Time series Multiple position Multiple z-sections April 2013 Deconvolution microscope Widefield PSF SVI Using maths to remove haze and blur April 2013 Deconvolution microscope 192 optical sections of a fruit fly embryo leg acquired in 0.4-micrometer z-axis steps with a widefield fluorescence microscope April 2013 Confocal laser scanning microscope sample 3D-reconstruction Z-series First confocals in the 50s First commercial system in 1987 3D pixel = voxel April 2013 Confocal laser scanning microscope Optical sections 3D-reconstruction Blue = DNA Green = Centrosome Red = Microtubules April 2013 April 2013 Resolution CLSM 128x128 256x256 512x512 1024x1024 April 2013 Confocal laser scanning microscope Deconvolution Set top Set bottom April 2013 Spinning disk microscope CLSM 30° turn is 1 image; 360° is 12 images ~ 2000 images/second. However, exposure time might be 100 milliseconds (quite common when using fluorescent proteins in living cells). April 2013 Spinning disk microscope widefield Spinning disk CLSM Z XY PSF Stefan Terjung, EMBL April 2013 Multi-photon laser scanning microscope April 2013 Multi-photon laser scanning microscope April 2013 Multi-photon laser scanning microscope Even scattered fluorescence photons are usefull in 2photon regime April 2013 Multi-photon laser scanning microscope • Penetration depth CLSM ~ 30-50 µm Use of pulsed infrared laser increases penetration depth to up to 1000 µm Used for imaging of thicker samples like mouse embryos, mammalian brain, intact nervous system, bone-marrow. April 2013 Multi-photon laser scanning microscope CLSM (50 µm) Mouse cortical pyramidal neurons expressing EYFP. Excitation using 920 nm. (Zeiss LSM 7 MP brochure) Transgenic mice expressing a mixture of different fluorescence proteins but individual neurons expres each a unique combination of this mixture (brainbow). Williams et al. (2010) J NeuroSci. 30: 11951 April 2013 Total internal reflection (TIRF) microscope April 2013 Total internal reflection (TIRF) microscope Advantages: + High signal to noise ratio + Very fast acquisition possible + Single molecule detection + Very good for studying vesicle-membrane fusion events and cell adhesion Disadvantages: - Only fluorescence directly at cover slip April 2013 Systems Advantages Disadvantages Deconvolution •High light efficiency •Very fast frame rates possible (CCD/sCMOS) •Low depth discrimination •Filterchanges usually slow •Results only after deconvolution CLSM •High resolution •Optical zoom •Optical sectioning •Low signal/noise •Relative slow acquisition (PMT) Spinning disk •Optical sectioning less than CLSM •Higher frame rate than CLSM (CCD/sCMOS) •Multichannel usually sequential •No optical zoom •Less depth discrimination than CLSM MP LSM •Optical sectioning without pinhole •High resolution •Deep penetration (NIR) •Only excitation at focal point •Expensive •One channel at a time •Relative slow acquisition (PMT) TIRF •High signal/noise •Very fast frame rates possible (CCD/sCMOS) •Single molecule detection •Only fluorescence directly at cover slip Structured illumination OptiGrid system QiOptiq April 2013 Structured illumination Grid is only in focus in the focal plane of the sample Three raw images are combined to one in which the grid lines and the out of focus signal have disappeared due to some claver calculations. Contrast and image sharpness are markedly improved Carl Zeiss MicroImaging, Thornwood, April 2013 Structured illumination Advantages + Cheap + Can be added to any fluorescence microscope + Results are directly visible Disadvantages - Slow No live cell imaging! - Need bright sample, increased bleaching April 2013 Light sheet microscopy Huisken J , Stainier D Y R Development 2009;136:1963-1975 April 2013 Light sheet microscopy Principle discribed in 1903, first system in 1993 by Voie et al. (J. Microsc. 170: 229–236. ) orthogonal-plane fluorescence optical sectioning (ORFOS) Selective plane illumination microscopy (SPIM) Digital scanned laser light sheet fluorescence microscopy (DSLM) Thin laser light sheet microscope Light sheet microscopy (TLSM) April 2013 Light sheet microscopy Reconstruction of Zebrafish Early Embryonic Development Keller et al (2010) Nature methods April 2013 Super resolution microscopy Imaging with a resolution below the diffraction limit of light Structured illumination microscopy (SIM) Photo-activation localization microscopy (PALM) Stochastic optical reconstruction microscopy (STORM) and 3D STORM Stimulated Emission Depletion microscopy (STED) Ground State Depletion microscopy (GSD) 4pi (taken of the market) April 2013 Structured illumination microscopy (SIM) April 2013 Structured illumination microscopy (SIM) CLSM SIM XY-resolution 100 nm Z- resolution 200-300 nm • Still diffraction limited • Normal dyes • Two fold resolution improvement April 2013 Structured illumination microscopy (SIM) DeltaVision | OMX 3D-SIM™ Super-Resolution Imaging April 2013 Stimulated Emission Depletion microscopy (STED) Fluorescence is completely suppressed by stimulated emission process. This ring-shaped pulse effectively provides a tiny aperture April 2013 Stimulated Emission Depletion microscopy (STED) Sieber et al. Science 317: 1072-1076 April 2013 Stimulated Emission Depletion microscopy (STED) Bückers et al., Opt. Express 19 (2011): 3130 - 3143 April 2013 Ground state depletion microscopy photobleaching April 2013 Ground state depletion microscopy photobleaching April 2013 Ground state depletion microscopy Dark state of a fluorophore; higher energy state that does not omit light 500 frames; 22 frames/s Q-dots CellR system Straatman Olsen and McKenzie (2010) A dark excited state of fluorescent protein chromophores, considered as Brooker dyes. Chemical Physics Letters 492: 150-156 April 2013 Ground state depletion microscopy Ptk2-cells. Anti-NUP153/Alexa FLUOR 532 and anti-β-tubulin/Alexa FLUOR 488 Wernher Fouquet, Leica Microsystems, Anna Szymborsak and Jan Ellenberg, EMBL, Heidelberg, Germany. According to Leica website: Acquisition time 2-10 minutes Maximum resolution down to 20 nm Standard fluorochromes can be used – no need to change your protocols April 2013 PALM/STORM PALM: use photo-activatable dyes (e.g., paGFP) STORM: need photo-switchable fluorochromes Imaging laser (657 nm) Activator Cy3 Cy5 Reporter 6000 photons Activation Cy3 Cy5 Cy3 Cy5 Activation laser (532 nm) April 2013 PALM/STORM/GSD Bright field microscope April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD April 2013 PALM/STORM/GSD Brightfield STORM April 2013 █ Cy3 / Alexa 647: Clathrin █ Cy2 / Alexa 647: Microtubule 5 μm Bates et al, Science 317, 1749 – 1753 (2007) 1 μm And there is now also 3D STORM 200 nm Resolvable volumes obtained with current commercial super-resolution microscopes © 2010 Schermelleh et al. /GSD Schermelleh L et al. J Cell Biol doi:10.1083/jcb.201002018 April 2013 Whole animal imaging systems Multispectral imaging March 2012 Whole animal imaging systems A; Original RGB image C; FITC D; TRITC E; Cy3.5 F; Food G; Skin autofluorescence H; Merge Levenson and Mansfield, Cytometry A 2006 March 2012 http://zeiss-campus.magnet.fsu.edu/tutorials/basics/axioobserver/index.html Light sheet microscopy Reconstruction of Zebrafish Early Embryonic Development H2B-eGFP mRNA injected in one-cell stage Total about 400,000 images per embryo Keller et al (2008) Science 322 April 2013