S1007 Doing structural biology with the electron microscope Lecture 2: Sample preparation 2 Samples 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) 3 Interaction of electrons with matter Mean free path 4 Interaction of electrons with matter SEM TEM 5 Interaction of electrons with matter SEM TEM 6 Scanning electron microscopy 7 Transmission electron microscopy BF image Objective aperture - difference in intensity in two adjacent area - Transmitted and diffracted waves travel through different distances Amplitude contrast Phase contrast 8 Sample preparation techniques Thin section methods Heavy metal staining and shadowing Plunge freezing High pressure freezing 9 Thin section methods Chemical fixation (formaldehyd, glutaraldehyde, osmium tetraoxide) Dehydration (EtOH, aceton) Plastic embedding Sectioning 10 Thin section methods Chemical fixation - 2% solution in buffer or water - variable duration – sample thickness (2-24hours) 11 Thin section methods - high vacuum in the miscorscope - EtOH, aceton - succesive increase of dehyd. agent concentration 30% aceton - 15 mins 50% aceton - 15 mins 70% aceton - 15 mins 90% aceton - 15 mins 100% aceton - 3 changes Drawbacks: - contraction of protein lipids - sample shrinking up to 40% - fromation of various artefacts Dehydration 12 Thin section methods Resin infiltration: 2:1 mix of propylene oxide:resin (1h) 1:1 mix of propylene oxide:resin (1h) 1:2 mix of propylene oxide:resin (1h) 100% resin overnight Polymerization: 12-24 hours at 60-70C Resin embedding 13 Thin section methods Sectioning 14 Thin section methods Mechanical sectioning for TEM 15 Thin section methods Mechanical sectioning/block-face for SEM 16 Thin section methods Focused ion beam block-face for SEM 17 Heavy metal staining Stains: uranyl acetate (pH=4) uranyl formate (pH=4) ammonium molybdenate (pH=7) phosphorus thungstanate (pH=7) Negative staining 18 Heavy metal staining Pros: quick sample screening high amplitude contrast less prone to beam damage Cons: limited resolution (20A) flattening artefacts denaturation of proteins Negative staining 19 Heavy metal staining Negative staining 20 Heavy metal staining - DNA visualization Metal shadowing 21 - non-physiological conditions during sample preparationd - artefacts (changes in cell structure, depression of proteins) - extremely toxic chemicals used during sample prep (OsO4) - obtainable level of detail limited + high signal to noise + low dose sensitivity + robust (easy sample handling) Thin section methods and heavy metal staining 22 Plunge freezing    Rapid immersion of buffered sample into cryogen  Cryogens: liquid ethane  ethane:propane mixture  Vitrification has to be fast ~1000 K/s   Possible only for samples with thickness ~<10um  => amorphous ice  => thin layer (200-600nm) 23 Plunge freezing 3-4ul 0.1-1mg/ml for purified protein complexes OD~0.5 for bacteria 24 Plunge freezing    Sample frozen in hydrated state   Amorphous ice   Sample has to be kept at temperatures  above devitrification point (~-135C)   Internal structures can be visualized   High resolution information is retained  Possible problems: ice thickness  hexagonal ice, cubic ice 25 Plunge freezing Extrusion of particles from thin ice Denaturation at air water interface 26 Plunge freezing    Cons:   Low signal to noise   Prone to radiation damage   More delicate sample handling required  27 High pressure freezing    Vitrification of samples with thickness  10 – 200 um   2000 bars, liquid nitrogen, 20 ms  Eukaryotic cells, tissues,...  Coupled with freeze substitution and  sample thinning using cryo-ultramicrotomy, ultramicrotomy, or focused ion beam milling 28 High pressure freezing  Freeze substitution  Reduce ultra-structure changes at room  due to dehydration as seen at ambient  temperature   Dehydration at temperatures belo -70C  (aceton typically -90C)  Fixatives are evenly distributed before  cross-linking at ambient temperature  Resin embedding for ultramicrotomy  at room temperature 29 Focus ion beam milling 30 Focus ion beam milling 31 Focus ion beam milling Středoevropský technologický institut c/o Masarykova univerzita Žerotínovo nám. 9 601 77 Brno, Česká republika www.ceitec.cz | info@ceitec.cz