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Liisa Hirvonen edited section_Method_This_investigation_was__.tex
almost 9 years ago
Commit id: 326983246bcdba0426904781c40b2d32268e5dfc
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This investigation was performed using a dual mode cooled Hamamatsu C1790-13 EBCCD comprised of 512$\times$512 pixels where each pixel is 24$\times$24μm in size. The photocathode in the EBCCD is a GaAsP plate with an approximate quantum efficiency of 50\% when illuminated with 520nm photons. Upon liberation from the photocathode, the photoelectrons are accelerated across a potential difference into a back-thinned CCD. The vacuum chamber has an aluminium sheet 1.3mm from the cathode to protect the back-thinned CCD (from what?). During data acquisition, the EBCCD was cooled to an operation temperature of -15$^\circ$C and the EBCCD read-out was operated using the HiPic 7.1.0 software package from Hamamatsu, which acquired data using an exposure time of 10μs and a Super-High (EB?) gain.
The EBCCD was attached to the output port of an inverted Nikon Eclipse TE2000-E microscope. For the biological cell sample imaging, the microscope was used with a 100$\times$ 1.4NA air objective (Nikon) and for the 1951 USAF resolution test chart, the microscope was used with a 4$\times$ 0.13NA air objective (also Nikon). 2,000 frames were collected for the biological cell sample and 30,000 frames were collected for the USAF test pattern. The 10$\mu$s exposure frames contained single photon events and were processed with the thunderSTORM superresolution imaging plug-in for ImageJ, whose settings are explored in this paper. Due to computational memory restraints, the USAF test pattern data was processed in six 5,000 image stacks. The data is first processed to detect the photon events from the noise background, before implementing a localization algorithm to detect the centre of the photon event. Subsequently, a sub-pixel localization algorithm is used on the data to fragment the pixels into 13×13 grids to allow the centre of photon events to be located with greater resolution.