Adobe Systems Adobe Systems > Electron microscopy InnoCore project Jiri Novacek ‹#› > > Sylabus n Lecture 1: Applications of electron microscopy in life- science research n Lecture 2: Transmission electron microscope, cryo-electron microscopy, principles of image formation n Lecture 3: Data alignment in 2D, techniques for 3D model determination in cryo-EM ‹#› > > Sylabus n Lecture 1: Applications of electron microscopy in life- science research n Lecture 2: Transmission electron microscope, cryo-electron microscopy, principles of image formation n Lecture 3: Data alignment in 2D, techniques for 3D model determination in cryo-EM ‹#› > > Electron rest mass: 9.109 e-31 kg charge: -1.61 e-19 C spin: 1/2 Electron in electric and magnetic field (Lorentz force) ‹#› > > Electron Dual character of electron Acceleration Voltage [kV] Non-relativistic wavelength [pm] Relativistic wavelength [pm] 2 27.35 27.32 20 8.65 8.57 100 3.87 3.69 200 2.73 2.50 300 2.23 1.96 Abbe diffraction limit ‹#› > > Scales in electron microscopy 1 mm 1 um 1 nm Tick (ESEM) Plant cell (TEM) Plant (SEM) Bacteria (SEM) Virus (TEM) Bacteriophage (TEM) RNA polymerase (TEM) ‹#› > > Applications in life-sciences n SEM imaging n Block face imaging nStructural Biology cryo-EM n Cellular cryo-EM techniques ‹#› > > SEM imaging Pros: - imaging of sample morphology at significant scale difference(1mm - 10nm) - fast sample preparation Cons: - non-native (sample dehydrated) Sample preparation: - air drying - metal sputtering (Pt, Au, Ir) ‹#› > > SEM imaging Pros: - imaging of sample morphology at significant scale difference(1mm - 10nm) - fast sample preparation Cons: - non-native (sample dehydrated) Sample preparation: - freezing into LN2 - sublimation - metal sputtering (Pt, Au, Ir) ‹#› > > SEM imaging Pros: - imaging of sample morphology at significant scale difference(1mm - 10nm) - fast sample preparation Cons: - non-native (sample dehydrated) Sample preparation: - chemical fixation - contrasting (Pt,U) - dehydration (EtOH,aceton,HMDS) - critical point drying - metal sputtering (Pt, Au, Ir) ‹#› > > Block face imaging Pros: - 3D volume reconstruction at ultrastructural level of detail - high signal to noise - low dose sensitivity - robust (easy sample handling) Cons: - non-physiological conditions during sample prep - artefacts (changes in cell structure, depression of proteins) - extremely toxic chemicals (OsO4) - attainable level of detail limited ‹#› > > Block face imaging Pros: - 3D volume reconstruction at ultrastructural level of detail - high signal to noise - low dose sensitivity - robust sample preparation Cons: - non-physiological conditions during sample prep - artefacts (changes in cell structure, depression of proteins) - extremely toxic chemicals (OsO4) - attainable level of detail limited Sample preparation 1: - formaldehyde, glutaraldehyde - chemical fixation - ~2% solution in water or buffer - variable duration – 2-24 hours (sample thickness) - contrasting (OsO4, UAc, Pb) ‹#› > > Block face imaging Pros: - 3D volume reconstruction at ultrastructural level of detail - high signal to noise - low dose sensitivity - robust sample preparation Cons: - non-physiological conditions during sample prep - artefacts (changes in cell structure, depression of proteins) - extremely toxic chemicals (OsO4) - attainable level of detail limited Sample preparation 2: Dehydration – EtOH or aceton series (30% for 15mins, 50% for 15min, 70% for 15mins, 90% for 15mins, 100% - 3x) - shrinking of protein and lipids - sample shrinking up to 40% - formation of various artefacts Resin embedding – resin infiltration ( 2:1 propylen oxide: resin for 1h, 1:1 for 1h, 1:2 for 1h, 100% resin overnight - polymerazation 24-72h at 60-70C ‹#› > > > Block face imaging Mechanical sectioning for TEM ‹#› > > > Block face imaging Mechanical sectioning for TEM - 50 – 70 nm thick sections - high-resolution imaging in TEM (tomography) - 3D volume reconstruction - resolution limited by sample preparation - staining with EM contrasting agents (nanoparticles) or fluorescent markers (CLEM) for targetting NIH el. mic. facility ‹#› > > > Block face imaging Mechanical or FIB sectioning for SEM - detection of back scattered electrons - mechanical sectioning either inside or outside SEM - FIB sectioning (10nm) - FIB-SEM tomography – correlative studies limited ‹#› > > Block face imaging ‹#› > > Heavy metal staining (negative staining) Stains: uranyl acetate (pH=4) uranyl formate (pH=4) ammonium molybdenate (pH=7) phosphorus thungstanate (pH=7) ‹#› > > Heavy metal staining (negative staining) Pros: - quick sample screening - high contrast - less prone to beam damage ‹#› > > Heavy metal staining (negative staining) Pros: - quick sample screening - high contrast - less prone to beam damage Cons: - limited resolution (20A) - flattening artefacts - denaturation of proteins ‹#› > > Cryo-EM techniques Plunge freezing: - rapid immersion of buffered sample into cryogen (liquid ethane, ethane:propane mix) - vitrification has to be fast 10e4-10e5 K/s - available only for samples ~<10um thick High pressure freezing - sample thickness <200um - freezing with liquid nitrogen - 2000 bars, 20 ms ‹#› > > Cryo-EM techniques > 3-4ul 0.1-1mg/ml for purified protein complexes OD~0.5 for bacteria > Plunge freezing ‹#› > > Cellular cryo-EM techniques > High pressure freezing, freeze substitution Freeze substitution - reduction of ultrastructure changes compared to dehydration at ambient temperature - dehydration at temperatures <-70C (aceton typically -90C) - fixatives are evenly distributed before cross-linking at ambient temperature - resin embedding for ultramicrotomy at room temp. www.leica-microsystems.com Yamada et al. JMM 2010 ‹#› > > > Cellular cryo-EM techniques CEMOVIS – cryo-EM of vitrous sections - no chemical fixation, dehydration or contrasting - low contrast - preservation of the sample in near-native conditions - mechanical sectioning by ultramicrotome at LN2 conditions - sectioning artefacts Al-Amoudi et al. EMBO J 2004 Al-Amoudi et al. JSB 2005 ‹#› > > Cellular cryo-EM techniques > Focused ion beam milling of cellular lamellas ‹#› > > Cellular cryo-EM techniques ‹#› > > Cellular cryo-EM techniques Vaccinia virus inside cell HeLa cells Pavel Plevka group - no chemical fixation, no heavy atom enhancement - true, near-native representation of the cellular interior - low contrast ‹#› Thank you for attention jiri.novacek@ceitec.muni.cz