Superior performance with sCMOS over EMCCD in super-resolution optical fluctuation imaging.
Super-resolution optical fluctuation imaging (SOFI) is a fast and low-cost live-cell optical nanoscopy for extracting subdiffraction information from the statistics of fluorescence intensity fluctuation. As SOFI is based on the fluctuation statistics, rather than the detection of single molecules, it poses unique requirements for imaging detectors, which still lack a systematic evaluation. Here, we analyze the influences of pixel sizes, frame rates, noise levels, and different gains in SOFI with simulations and experimental tests. Our analysis shows that the smaller pixel size and faster readout speed of scientific-grade complementary metal oxide semiconductor (sCMOS) enables SOFI to achieve high spatiotemporal resolution with a large field-of-view, which is especially beneficial for live-cell super-resolution imaging. Overall, as the performance of SOFI is relatively insensitive to the signal-to-noise ratio (SNR), the gain in pixel size and readout speed exceeds the loss in SNR, indicating sCMOS is superior to electron multiplying charge coupled device in context to SOFI in many cases. Super-resolution imaging of cellular microtubule structures with high-order SOFI is experimentally demonstrated at large field-of-view, taking advantage of the large pixel number and fast frame rate of sCMOS cameras.
Peking University, College of Engineering, Department of Biomedical Engineering, No. 5 Yiheyuan Road Haidian District, Beijing 100871, ChinabPeking University, State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of.
Xuanze
X
Chen
Peking University, College of Engineering, Department of Biomedical Engineering, No. 5 Yiheyuan Road Haidian District, Beijing 100871, China.
Zhiping
Z
Zeng
Peking University, State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, No. 5 Yiheyuan Road Haidian District, Beijing 100871, China.
Rongqin
R
Li
Peking University, State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, No. 5 Yiheyuan Road Haidian District, Beijing 100871, China.
Boxin
B
Xue
Peking University, College of Engineering, Department of Biomedical Engineering, No. 5 Yiheyuan Road Haidian District, Beijing 100871, China.
Peng
P
Xi
Peking University, State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, No. 5 Yiheyuan Road Haidian District, Beijing 100871, China.
Yujie
Y
Sun
D003198:Computer Simulation; D008856:Microscopy, Fluorescence / Q000592:standards; D061848:Optical Imaging / Q000379:methods*; D012666:Semiconductors / Q000592:standards*; D059629:Signal-To-Noise Ratio