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Neurons are plated on gold finder grids in a cell culture dish and treated with unlabeled or Alexa Fluor 488 labeled Ab42 oligomers for 3 days. LysoTracker Red is added to label lysosomes and gold fiducials added to aid tomogram alignment at later processing stages. The grids with neurons are then plunge frozen in liquid ethane and screened using cryogenic widefield microscopy to select areas to analyze further using cryo-SIM to image Ab42 oligomer and lysosome fluorescence followed by cryo X-ray microscopy to view the cellular ultrastructure. Finally, various data processing stages are carried out to generate three dimensional cellular maps and quantify the lysosomal changes. Created withBiorender.com.

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