Photon counting imaging was performed with a dual mode cooled Hamamatsu C1790-13 EBCCD, with \(512\times512\) pixels and \(24\times24\) \(\mu\)m pixel size. The EBCCD was cooled to -15\(^\circ\)C, and HiPic 7.1.0 software was used for image acquisition with 10 \(\mu\)s exposure time and super-high amplifier gain. The EBCCD was attached to the output port of an inverted Nikon Eclipse TE2000-U microscope, as schematically illustrated in Fig \ref{fig1}a. For transmission imaging of a 1951 USAF resolution test chart (Fig \ref{fig1}b), the microscope was used with a 4\(\times\) 0.13NA air objective (Nikon) and a halogen lamp. For epifluorescence imaging, a cell sample (FluoCells Prepared Slide #1, Molecular Probes) was excited with a pulsed 467 nm diode laser (Hamamatsu PLP-10) and imaged with a 100\(\times\) 1.4NA oil objective (Nikon). The illumination intensity was adjusted such that single photon events could be observed (Fig \ref{fig1}c,d).
The frames containing single photon events were processed with ThunderSTORM \cite{Ovesny2014} superresolution imaging plug-in for ImageJ. Due to memory limitations, the USAF test chart data was processed in 6\(\times\)5,000 and the cell data in 3\(\times\)2,000 image stacks. The software first detects the events from the noise background, and an approximate localisation algorithm locates the center pixel of each event. A sub-pixel localisation algorithm then calculates the center of the events with greater resolution.
The software camera parameters were set to 80.0 nm pixel size and 36 photoelectrons per A/D count. The base level varied between image stacks due to fluctuations in the EBCCD temperature, and was set to the average minimum grey value for the image stack in the range of 100-140 A/D counts. A wavelet (b-spline) image filter was applied with order of 3 and scale of 2.0. For the approximate localisation of the events, the centroid of connected components method was used with a peak intensity threshold (PIT) of 2*std(Wave.F1) for the USAF test chart data, and a PIT of 1.5*std(Wave.F1) for cell data, with the watershed algorithm enabled for all data.
All sub-pixel localisation methods offered by ThunderSTORM (Maximum Likelihood (ML) and Least squares (LS) fitting with both Gaussian (G) and Integrated Gaussian (IG) point-spread function (PSF), Centroid of local neighborhood, Radial Symmetry) were tested, with fitting parameters optimised for maximum photon count and minimum likelihood of false photon event recognition.
For the best results, ML fitting was used with a Gaussian PSF, with standard deviation (SD) set to 1.0 pixels. For fast processing with adequate results the PSF fitting radius was set to 2 pixels, and for optimal photon detection and separation of overlapping events the radius was set to 7 pixels. Multiple-emitter fitting analysis (MFA) was tested with a maximum of 2 molecules per fitting region with a model selection threshold (p-value) of 10\(^{-6}\). When MFA was enabled, ThunderSTORM’s “remove duplicates” post-processing tool was applied with a distance threshold of 160 nm, and “intensity\(>\)4000” filter was applied to the USAF data and “intensity\(>\)3000” filter to the cell data.
Results are also shown for LS fitting method with an IG PSF with a 3 pixel fitting radius and 1.6 pixel SD, and a radial symmetry localisation method with 2 pixel estimation radius.