biostudies-literatureHari SDutch Research Council (NWO)ZonMwNanoNextNL, a consortium of the Dutch government and 130 public and private partners.20185https://www.ebi.ac.uk/biostudies/studies/S-EPMC9684522biostudies-literatureUnknown12(1)Super-resolution fluorescence microscopy can be achieved by image reconstruction after spatially patterned illumination or sequential photo-switching and read-out. Reconstruction algorithms and microscope performance are typically tested using simulated image data, due to a lack of strategies to pattern complex fluorescent patterns with nanoscale dimension control. Here, we report direct electron-beam patterning of fluorescence nanopatterns as calibration standards for super-resolution fluorescence. Patterned regions are identified with both electron microscopy and fluorescence labelling of choice, allowing precise correlation of predefined pattern dimensions, a posteriori obtained electron images, and reconstructed super-resolution images.Scientific reportsElectron-beam patterned calibration structures for structured illumination microscopy.PMC9684522435002021711.016.003ECHO-711.016.003Hoogenboom JPVos YFloris CHoutsmuller ABStallinga SSlotman JAHari SHagen CWvan Cappellen WAfalseElectron-beam patterned calibration structures for structured illumination microscopy.Super-resolution fluorescence microscopy can be achieved by image reconstruction after spatially patterned illumination or sequential photo-switching and read-out. Reconstruction algorithms and microscope performance are typically tested using simulated image data, due to a lack of strategies to pattern complex fluorescent patterns with nanoscale dimension control. Here, we report direct electron-beam patterning of fluorescence nanopatterns as calibration standards for super-resolution fluorescence. Patterned regions are identified with both electron microscopy and fluorescence labelling of choice, allowing precise correlation of predefined pattern dimensions, a posteriori obtained electron images, and reconstructed super-resolution images.2022-01-01T00:00:00Z2022 Nov2025-04-18T14:59:37.477Z2025-04-07T01:26:14.385ZS-EPMC96845223641842010.1038/s41598-022-24502-0