TheLeeLab, at the University of Cambridge, focuses its research on developing biophysical tools to answer fundamental biological questions, primarily using single-molecule fluorescence imaging techniques. Recently the group has established single-molecule spectroscopic imaging, facilitating local hydrophobicity mapping (1), as well as implemented cutting-edge point-spread function engineering for large-volume single-particle tracking in live T cells (2).
As with all single-molecule techniques, sensitivity is vital as the photon budget of individual fluorophores can be limited. For single-particle tracking applications, detecting more photons allows for longer trajectories to be recorded and more robust statistical analysis to be conducted.
Additionally, the motion of a fluorophore must be adequately sampled in order to accurately record its behaviour. For fast-moving targets, such as many cytoplasmic and nucleic proteins, short exposure times are required and thus the emission is spread over a greater number of frames.
In order to achieve live single-particle tracking of fast-moving targets, both high sensitivity and fast acquisition rates are vital. Although EMCCDs have previously been used to achieve both of these factors, high speed came at the cost of a much reduced field of view, which made data collection inefficient.
The high quantum efficiency and low noise of the Prime BSI sCMOS combines EMCCD-level sensitivity with a fast acquisition rate and much larger field of view. The Prime BSI speeds up data collection as multiple cells can be imaged within the same field of view at >100 fps with high contrast.