Table 2: A comparison of the different approaches for time-resolved cryoEM according by their technical properties.

Publications that propose several methods are split into separate rows. N / S (not stated) means that the publication does not specify a value.

Studied system	trEM approach	Time resolution calibration	Time adjustment	Needed equipment / Availability
Dissociation of actomyosin75	Flash photolysis of caged components before freezing	Actomyosin-S1 dissociation	Falling height between flash lamp and liquid ethane container	Flash lamp and photo-electric trigger
NaCl induced changes of Erythrocytes68	Gas-assisted spray of one reactant on blotted grid	N / S	varying plunging time (velocity or distance)	widely available components, legacy atomizer
Dissociation of actomyosin73	Voltage-assisted spray of one reactant on blotted grid	N / S	Plunge speed (via plunging pressure), plunge time (via plunging distance), intermediate stop	Syringe pump, high voltage DC converter and custom-built machine with widely available components
	Voltage-assisted spray of reactants mixed in Berger ball	N / S	(as above)	(as above)
Mixed particle colocalisation50	Voltage-assisted spray	N / S	N / S	(as above)
Microfluidic chip mixing channel82	Voltage-assisted spray of one reactant on blotted grid	N / S	N / S	(as above)
Microfluidic chip sprayer nozzle47	Gas-assisted spray of reactants mixed in microfluidic silicon chip (butterfly mixers)	N / S	Different chips with varying delay length	Microfabrication equipment
70S Ribosome tRNA retro-translocation43	characterisation experiments of device above	measurement and CFD simulation of mixing index and residence time distribution	(as above)	(as above)
Sprayed apoferritin81	Gas-assisted spray of reactants mixed in microfluidic silicon chip, improved spray	(as above)	(as above)	3D MEMS fabrication equipment
Mixed particle colocalisation50	manual trEM	stopwatch	longer incubation in the minute range	controlled environment vitrification system/ standard EM equipment
Microfluidic chip mixing channel82	Gas-assisted spray from microfluidic silicon chip	N / S	N / S	Microfabrication equipment
Mixed particle colocalization and dissociation of actomyosin70	Voltage-assisted spray of reactants mixed in T-mixers	N / S	Residence time and plunge speed (via plunging pressure)	widely available components
	Voltage-assisted spray of one reactant on blotted grid	N / S	Plunge speed (via plunging pressure)	(as above)
70S ribosome association, MthK Ca2+ binding, RNA pol DNA association, GTP hydrolysis dependent rearrangement of dynamin69	Inkjet application of one reactant onto the other	70S ribosome assembly was monitored	varying plunging time (velocity or distance) (90-500ms tested so far	proprietary device
RecA filament growth71	Gas-assisted spray of reactants mixed in microfluidic silicon chip	Time dependent RecA + ssDNA/ATPyS filament growth	Different chips with varying delay length	Microfabrication equipment
Actomyosin dissociation113	Gas-assisted spray of one reactant on blotted grid	Actomyosin dissociation, Plunge speed measurement + liquid flow rate + droplet	plunge stop	Microfabrication equipment
	Gas-assisted spray of reactants mixed in a T-mixer	(as above)	varying delay line inner diameter	(as above)
GroEL disassembly upon electron-beam damage78	Rapid melting and revitrification inside the electron microscope	time series of refraction at ice and gold film in EM	Varying laser pulse times	Customization of the TEM with lasers for electron beam pulsing and heating
Apoferritin revitrification114	(as above)	(as above)	(as above)	(as above)