Sample preparation techniques Thin section methods Heavy metal staining and shadowing Plunge freezing High pressure freezing Focus ion beam milling Interaction of electrons with specimen Williams et al., TEM, Springer Interaction of electrons with specimen Williams et al., TEM, Springer SEM TEM Contrast in EM images Amplitude contrast BF image Objective aperture C= ( I 2−I1 ) I1 = ΔI I1 - difference in intensity in two adjacent area Contrast in EM images Phase contrast - Transmitted and diffracted waves travel through different distances Thin sectioning methods Chemical fixation (formaldehyd, glutaraldehyde, osmium tetraoxide) Dehydration (EtOH, aceton) Plastic embedding Sectioning Thin section methods Chemical fixation - formaldehyd - 2% solution in buffer or water - variable duration – sample thickness (2-24hours) Thin section methods Dehydration - 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 Thin section methods Resin embedding 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 Thin section methods Sectioning Heavy metal staining and shadowing Negative staining Stains: uranyl acetate (pH=4) uranyl formate (pH=4) ammonium molybdenate (pH=7) phosphorus thungstanate (pH=7) Heavy metal staining and shadowing Negative staining Pros: quick sample screening high amplitude contrast less prone to beam damage Heavy metal staining and shadowing Negative staining Pros: quick sample screening high amplitude contrast less prone to beam damage Cons: limited resolution (20A) flattening artefacts denaturation of proteins Heavy metal staining and shadowing Metal shadowing - DNA visualization Hashimoto et al., NMSB 2010 Plunge freezing - rapid immersion of buffered sample into cryogen - cryogens: liquid ethane, ethane:propane mixture -vitrification has to be fast ~10000 K/s => amorphous ice => thin layer (200-600nm) Plunge freezing Cheng et al. Cur. Microscopy 2010 - rapid immersion of buffered sample into cryogen - cryogens: liquid ethane, ethane:propane mixture -vitrification has to be fast ~10000 K/s => amorphous ice => thin layer (200-600nm) Pros: - sample in frozen hydrated state (native) - internal structures can be visualized - high resolution information preserved Plunge freezing - rapid immersion of buffered sample into cryogen - cryogens: liquid ethane, ethane:propane mixture -vitrification has to be fast ~10000 K/s => amorphous ice => thin layer (200-600nm) Pros: - sample in frozen hydrated state (native) - internal structures can be visualized - high resolution information preserved Cons: - low signal to noise - prone to radiation damage - sample handling more difficult Extrusion of particles from thin ice Denaturation at air water interface Plunge freezing - rapid immersion of buffered sample into cryogen - cryogens: liquid ethane, ethane:propane mixture -vitrification has to be fast ~10000 K/s => amorphous ice => thin layer (200-600nm) Pros: - sample in frozen hydrated state (native) - internal structures can be visualized - high resolution information preserved Extrusion of particles from thin ice Denaturation at air water interface High pressure freezing