Effects of fixed pattern noise on single molecule localization microscopy

The newly developed scientific complementary metal oxide semiconductor (sCMOS) cameras are capable of realizing fast single molecule localization microscopy without sacrificing field-of-view, benefiting from their readout speed which is significantly higher than that of conventional charge-coupled device (CCD) cameras. However, the poor image uniformity (suffered from fixed pattern noise, FPN) is a major obstruction for widespread use of sCMOS cameras in single molecule localization microscopy. Here we present a quantitative investigation on the effects of FPN on single molecule localization microscopy via localization precision and localization bias. We found that FPN leads to almost no effect on localization precision, but introduces a certain amount of localization bias. However, for a commercial Hamamatsu Flash 4.0 sCMOS camera, such localization bias is usually <2 nm and thus can be neglected for most localization microscopy experiments. This study addresses the FPN concern which worries researchers, and thus will promote the application of sCMOS cameras in single molecule localization microscopy.