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\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$m pixel size.The photocathode in the EBCCD is a GaAsP plate with an approximate quantum efficiency of 50\% at 520~nm.  Photoelectrons liberated from the GaAsP  photocathode are accelerated across a potential difference into a back-thinned CCD. The EBCCD was cooled to an operation temperature of -15$^\circ$C. -15$^\circ$C, and  HiPic 7.1.0 software was used for image acquisition, acquisition  with 10$\mu$s  exposure timeof 10$\mu$s  and super-high amplifier gain.The EBCCD was attached to the output port of an inverted Nikon Eclipse TE2000-E microscope, see Fig~\ref{fig1}a. For the 1951 USAF resolution test chart (Fig~\ref{fig1}b), the microscope was used with a 4$\times$ 0.13NA air objective (Nikon). A fluorescent cell sample (FluoCells Prepared Slide \#1, Molecular Probes) was 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).    \begin{figure}[tbp]  \centerline{\includegraphics[width=1\columnwidth]{fig1}}  \caption{\label{fig1} (a) Schematic diagram of the data acquisition setup. (b) Total imaged area of USAF test pattern. (c) A frame of raw data with single-photon events. (d) 3D representation of (c).}  \end{figure}  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 to computational memory restraints, the USAF test pattern data was processed in six 5,000 image stacks. The data is software  first processed to detect detects  thephoton  events from the noise background, before a an approximate  localization algorithm locates theapproximate  centre pixel  of the photon each  event. Subsequently, a sub-pixel localization algorithm is used to calculate calculates  the centre of photon 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.  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, To achieve good photon detection  and was set to adequate resolution fast,  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 data can be processed with Rapid Estimation (RE) settings using Maximum Likelihood (ML) fitting with Gaussian point-spread function (PSF)  with a b-spline order fitting radius  of 3 2 pixels  and a b-spline scale standard deviation (SD)  of 2.0. For 1.0 pixels. To achieve optimal photon detection,  the approximate localization algorithms, centroid resolution  of connected components (CoCC) was used with overlapping events and  a peak intensity threshold (PIT) of 2*std(Wave.F1) for lower FPN, the Maximum Resolution (MR) settings can be applied to  theUSAF  data, and however this produces  a PIT of 1.5*std(Wave.F1) for cell data, substantial increase in processing time. For MR settings, Maximum Likelihood (ML) fitting method was used  with watershed algorithm enabled for all data. Gaussian PSF with 7 pixel fitting radius and SD of 1.0 pixels.  For the sub-pixel localization algorithms, integrated Gaussian (IG) PSF was set to a 3 pixel Multiple-emitter  fitting radius, a 1.6 pixel standard deviation (SD) and the least squares (LS) fitting method and radial symmetry analysis (MFA)  was applied tested  with MR settings and  a maximum of  2 pixel estimation radius. To achieve good photon detection molecules per fitting region  withadequate FPN in  a very short period model selection threshold (p-value)  of time, the data can be processed 10$^{-6}$. When MFA was enabled, ThunderSTORM's ``remove duplicates'' post-processing tool was applied  with the Rapid Estimation (RE) settings described below. To achieve optimal photon detection, the resolution a distance threshold  of overlapping events 160~nm,  and a lower FPN, the Maximum Resolution (MR) settings can be ``intensity$>$4000'' filter was  applied to the data, however this produces a substantial increase in processing time. For the RE processing approximate localization algorithms, Gaussian PSF was set to a 2 pixel fitting radius with a 1.0 pixel SD and the Maximum Likelihood (ML) fitting method, USAF data  and for the MR processing, Gaussian PSF was set ``intensity$>$3000'' filter  toa 7 pixel fitting radius with a 1.0 pixel SD with  the Maximum Likelihood (ML) fitting method. cell data.  When the multiple-emitter Least squares (LS)  fitting analysis (MFA) method  was applied in conjunction to the also tested with integrated Gaussian (IG)  PSFgaussian or IG algorithms, it was used  with a maximum of 1 molecule per 3 pixel  fitting region radius  and a model selection threshold (p-value) of 10$^{-6}$ for the RE processing, or a maximum of 2 molecules per fitting region 1.6 pixel SD,  and a model selection threshold (p-value) of 10$^{-6}$ for the MR processing. For USAF test pattern data processed using the MR settings with MFA enabled, ThunderSTORM's ``remove duplicates'' post-processing tool was applied radial symmetry localisation method  with a distance threshold of 160~nm and an ``intensity$>$4000'' filter was used. For cells data, the remove duplicates tool was also applied with a distance threshold of 160~nm but with an ``intensity$>$3000'' filter. No post-processing was required for data processed using RE settings. 2 pixel estimation radius.