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\section{Method}  \subsection{Data acquisition} Single photon data was acquired with a dual mode cooled Hamamatsu C1790-13 EBCCD, with 512$\times$512 pixels and 24$\times$24~$\mu$m pixel size. The EBCCD was cooled to an operation temperature of -15$^\circ$C, and HiPic 7.1.0 software was used for image acquisition with 10~$\mu$s exposure time and super-high amplifier gain. The EBCCD was attached to the output port of an inverted Nikon Eclipse TE2000-E microscope, see Fig~\ref{fig1}a. For transmission imaging of a 1951 USAF resolution test chart (Fig~\ref{fig1}b), the microscope was used with a 4$\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 event could be observed (Fig~\ref{fig1}c,d).    %\subsection{Data processing}  Single photon data was acquired with a dual mode cooled Hamamatsu C1790-13 EBCCD, with 512$\times$512 pixels and 24$\times$24~$\mu$m pixel size. Photoelectrons liberated from the GaAsP photocathode are accelerated across a potential difference into a back-thinned CCD. The EBCCD was cooled to an operation temperature of -15$^\circ$C, and HiPic 7.1.0 software was used for image acquisition with 10$\mu$s exposure time and super-high amplifier gain.  The EBCCD was attached to the output port of an inverted Nikon Eclipse TE2000-E microscope, see Fig~\ref{fig1}a. For transmission imaging of the 1951 USAF resolution test chart (Fig~\ref{fig1}b), the microscope was used with a 4$\times$ 0.13NA air objective (Nikon) and a halogen lamp. For epifluorescence imaging, a cell sample (FluoCells Prepared Slide \#1, Molecular Probes) was excited 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 event could be observed (Fig~\ref{fig1}c,d).    \subsection{Data processing} The frames containing single photon events were processed with the ThunderSTORM \cite{Ovesny2014} superresolution imaging plug-in for ImageJ. Due tocomputational  memory restraints, the USAFtest pattern  data was processed in six 5,000 6$\times$5,000 and the cell data in 3$\times$2,000  image stacks. stack.  The software first detects the events from the noise background, before and  an approximate localization algorithm locates the centre center  pixel of each event. Subsequently, a A  sub-pixel localization algorithm the  calculates the centre center  of the events with greater resolution. The software camera parameters were set to 80.0~nm pixel size and 36 photoelectrons per A/D count. The base level varied between image stacks due to fluctuations in the EBCCD temperature, and was set to the average minimum grey value for the image stack in the range of 100-140 A/D counts. A wavelet (b-spline) image filter was applied with order of 3 and scale of 2.0. For the approximate localization of the events, centroid of connected components method was used with a peak intensity threshold (PIT) of 2*std(Wave.F1) for the USAF data, and a PIT of 1.5*std(Wave.F1) for cell data, with watershed algorithm enabled for all data.  To achieve good photon detection and adequate resolution fast, the data can be processed with Rapid Estimation (RE) settings using Maximum Likelihood (ML) fitting was used  with Gaussian point-spread function (PSF) (PSF),  witha fitting radius of 2 pixels and  standard deviation (SD) of set to  1.0 pixels. To achieve For fast processing with adequate results the PSF fitting radius was set to 2 pixels, and for  optimal photon detection, the resolution detection and separation  of overlapping eventsand a lower FPN,  the Maximum Resolution (MR) settings can be applied to the data, however this produces a substantial increase in processing time. For MR settings, Maximum Likelihood (ML) fitting method radius  was used with Gaussian PSF with set to  7pixel fitting radius and SD of 1.0  pixels. Multiple-emitter fitting analysis (MFA) was tested withMR settings and  a maximum of 2 molecules per fitting region with a model selection threshold (p-value) of 10$^{-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. Least squares (LS) fitting method was also tested with integrated Gaussian (IG) PSF with 3 pixel fitting radius and 1.6 pixel SD, and radial symmetry localisation method with 2 pixel estimation radius.