Flow system and methods for digital counting

A method of stabilizing a droplet pattern comprising the following steps:

• 1) providing a pattern of hydrophilic features in or on a hydrophobic substrate, the hydrophobic substrate being embedded in a flow compartment comprising a separate inlet and outlet, the hydrophilic features configured to support a plurality of liquid nano-to-attoliter droplets, 2) allowing a liquid to enter the inlet and exit the outlet of the flow compartment to form a plurality of liquid nano-to-attoliter droplets having an aggregate liquid volume VDA with a corresponding aggregate molar amount of liquid nDA, 3) blocking the inlet and outlet to provide a closed environment in the flow compartment of volume VC, 4) establishing a gas phase seal by allowing evaporation from the droplets into gas to establish a saturation vapor pressure within the closed flow compartment, wherein the vapor pressure increase at saturation is less than nDART/VC, where T is the temperature and R is the universal gas constant, such that the droplets, now having a reduced volume due to the evaporation, remain stable.