version 1 - 2021-10-20

The objective of this research was to find the theoretical minimum uncertainty for single-molecule localizations using a single-photon avalanche diode (SPAD). The key questions here was whether or not the binary output of a SPAD impacts the theoretical minimum uncertainty for the SPAD image model, or whether it is identical to the previously derived theoretical minimum uncertainty for an image model using Poissonian photon statistics.

After having theoretically derived this minimum uncertainty and having verified this result using simulation data we concluded that the difference between the Poissonian and binary image model is largest when saturation occurs in the pixels. To test this we had to create saturation in an experimental setup.

To do this we have built a TIRF setup where we observed DNA-PAINT nanorulers using a SwissSPAD2. Using this setup we acquired one large dataset taken at the minimum possible exposure time of the SwissSPAD2: 10 us. We then used this dataset to aggregate multiple frames into a single image, thereby setting any pixel value above 1 to 1, hereby creating saturation.

The .tiff data in this data set shows images for 8 different numbers of aggregated frames, from 16 aggregated frames to 2048 aggregated frames.