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A NP can reside in any position relative to pixel arrangement (only 4 pixels shown for representational purposes) and being in the size range of 1 pixel, can confound results. Pixels will light up (give a signal, represented as yellow in figure) even if a portion of the NP falls within their “borders” (pixel sizes depend on objectives; in current work, size was calculated to be ~ 161.2 nm for a 40× oil immersion lens used in an Olympus IX 70 inverted microscope).
