Live-Cell CLEM of Subcellular Targets

Bacteriophage Transcytosis Provides a Mechanism To Cross Epithelial Cell Layers

“…The sample was en bloc stained with 2% (wt/vol) aqueous uranyl acetate using three microwave duty cycles (120 s on and 120 s off) at 100 W under vacuum and then rinsed five times with MilliQ water. Microwave-assisted dehydration was performed at atmospheric pressure using 150 W for 40 s per stage of a graduated series of ethanol (50%, 70%, 90%, 100%, and 100%) and propylene oxide (100% and 100%), and microwave-assisted resin infiltration was performed under vacuum at 250 W for 180 s per stage using a graduated series of Procure-Araldite (25%, 50%, 75%, 100%, and 100%) in propylene oxide before resin polymerization at 60°C for 48 h. The target depth within the target cell was then relocated within the resin block, using the procedure outlined previously ( 39 , 62 ). The resin block was trimmed and then sectioned using an Ultracut UCT ultramicrotome (Leica) equipped with a 45° diamond knife (Diatome) to cut serial sections ( n = 49; average thickness, 79 nm) for collection on nine separate 300-mesh hex thin-bar copper grids.…”

Towards Polarizing Correlative Light-Electron Microscopy (PCLEM).

“…Технология корреляционной световой и электронной микроскопии (Correlative Light-Electron Microscopy (CLEM)) представляет собой сравнительно новый метод измерений на фиксированных ультратонких срезах и живых объектах [1][2][3][4][5]. В идеализированной форме, схематически, его можно представить, как способ установления колокализации маркеров и зон интереса (ROI) в электронном микроскопе с помощью вдвигаемой в его камеру (или к ней присоединенной с окнапри наличии соответствующей оптики) головки оптического микроскопа с установленной на ней цифровой камерой.…”