page_title: Single-molecule FRET
section_title: Camera blurring
hierarchical_section_title: Single-molecule FRET / Data Analysis of Surface Immobilised smFRET / Camera blurring
caption_attribution_description: English: Camera blur simulated with an example 3-state smFRET using the postFRET simulator (two simulations). The signal to noise ratio is set at about 10 for both simulations. The simulation rate constants are listed on the left, a small part of the trajectory is shown in the middle, and the histogram is shown on the right.
context_page_description: Single molecule fluorescence resonance energy transfer is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected on a single molecule level. In contrast to "ensemble FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium. Heterogeneity among different molecules can also be observed.
context_section_description: The camera blurring signal comes from the discrete nature of the measurements. The emission signal has a mismatch with the real transition signal because one or both are stochastic (random). When a state transition happens between the readout of two emission reading intervals, the signal is the average of the two parts in the same measuring window, which then affects the state identification accuracy and eventually the rate constant analysis. This is less a problem when the measuring frequency is much faster than the transition rate but becomes a real problem when they approach each other (figure on the right). In order to reflect the camera blurring effect in the simulated smFRET trajectories, one must simulate the data in a higher time resolution (e.g. 10 times faster) than the data collection time then bin the data into the final trajectories. See GitHub (postFRET) for an example MATLAB codes to simulate smFRET time trajectories with a faster time resolution then bin it to the measuring time resolution (integration time) to simulate the camera blurring effect.
caption: Camera blur simulated with an example 3-state smFRET using the postFRET simulator[8] (two simulations). The signal to noise ratio is set at about 20 for both simulations. The time resolution is simulated at 1 ms then binned to 10 ms, the integration time of the simulated experiments. The simulation rate constants are listed on the left, a small part of the trajectory is shown in the middle (alternative colors represent different molecules), and the FRET histogram of all molecules (100) is shown on the right.
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