Angström Resolution with Flow Immunofluorescence Localization Microscopy (FILM)

We present flow immunofluorescence localization microscopy (FILM) as a novel paradigm for single-molecule localization microscopy (SMLM). FILM is based on continuous flow of standard fluorescent binders, including conventional antibodies or nanoscale probes, over a fixed sample along with gentle imaging-to-photobleaching of the stochastically bound probes with an LED on a widefield microscope setup. Gentle illumination yields 104-106 photons per fluorophore corresponding to localization precision at the few nanometer to [A]ngstrom scales. We employ FILM to localize diverse markers in cultured cells and immunological synapses down to 9 [A] resolution, and for sequential localization of multiple targets in tissue slices. Requiring only minimal hardware and off-the-shelf probes, FILM offers an accessible and versatile route for serial SMLM at molecular resolution.