Table 2. Comparison of three different CLEM techniques, highlighting some properties of each technique and the advantages and disadvantages.

For further comments see section III.3.

	Tokuyasu	HPF	Simple
Quality of live cell imaging	Good	Not optimal due to distance between cells and objective.	Good
Labels	Internalised markers and high efficiency Immunolabelling	Internalised markers and surface Immunolabelling with HM20	Only internalised markers
Time resolution	Seconds to minutes	4 seconds	Seconds to minutes
Processing	Technically challenging	Automated if using an AFS with FSP.	Simple
Retracing cells	Embossed coverslip	Finder grids or carbon coated finder pattern	Embossed coverslip
Sectioning	Cryo sectioning. Difficult to achieve serial sections	Standard diamond knife sectioning of resin block	Standard diamond knife sectioning of resin block
Preservation of ultrastructure	Good	Excellent	Good
Time to complete experiment	4-5 days	4-7 days	2-3 days
Pitfalls	Formvar film too thick, cryosectioning, immunolabelling	Making the live cell carrier, achieving a well frozen sample	Removal of the coverslip