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Comparison on single molecule microscopy imaging with EM-CCD and qCMOS® camera ORCA®-Quest
The method of live-cell single-molecule fluorescence imaging plays an important role on various life science research fields, and sensitivity and resolution of the detector is critical for the imaging performance.
In order to achieve the best performance of single molecule imaging, Dr. Jiachao Xu, from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, built a TIRF microscope and has been continuously improving it in past several years.
Recently, Dr. Xu compared the performance of two kinds of cameras, EM-CCD and qCMOS, in his experiment of single molecule fluorescence imaging of angiotensin II type 1 receptor (AT1R) in living cells.
The benefits of ORCA-Quest
Dr. Jiachao Xu’s experiment requires the detector comes with very low noise, so as the fluorescence of single molecules to be easily recognized from a relatively clean background.
In his experience, EM-CCD could offer a good signal to noise ratio. He also tried some other types of cameras before, but none could achieve similar performance.
But this time qCMOS shows excellent performance with a readout noise almost as low as EM-CCD’s, even at the fast mode (0.43e r.m.s.). Almost the same quantity of fluorescent single molecules could be seen from the image of qCMOS and EM-CCD.
In addition, qCMOS has a smaller pixel size of 4.6 ㎛, which means it could be used under some modes: 4.6 ㎛ (1×1 binning), 9.2 ㎛ (2×2 binning) and 18.4 ㎛ (4×4 binning) depending on imaging demands. For example, to balance between sensitivity and localization precision, Dr. Xu choosed 9.2 ㎛ (2×2 binning) setting for the best performance.
In summary, qCMOS shows comparable or even better performance compared with EM-CCD on single-molecule fluorescence imaging. Its advantages on sensitivity, speed and resolution could provide more choices for different scientific imaging applications.
Image example by ORCA-Quest
Sample：AT1R-EYFP
Exposure time：100 ms
Optics：TIRF
Objective lens : 100× NA 1.49
laser：488 nm 1 mW
About TIRFM and Dr Jiachao Xu’s research
Dr. Jiachao Xu is from Dr. Kangmin He’s laboratory. Their research is highly praised in the field. The research method of real-time live-cell single particle tracking using TIRFM is "exciting, which will promote many peers to carry out similar research".
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