A single fluorescent molecule is imaged with a point-spread function of standard…
PHYSICS · Multi-part · Fermi University Physics · Optics & Photonics · Fourier optics and image formation · Fourier optics and image formation
What an Instrument Can See, Ch Seeing what the transfer function forbids: phase contrast and super-resolution
Problem
A single fluorescent molecule is imaged with a point-spread function of standard deviation σPSF=130 nm. In one camera frame, 850 photons from that molecule are detected above background.
((a)) Estimate the theoretical (centroid) localisation precision for this frame.
((b)) Background counts and finite pixel size typically inflate the theoretical value by about a factor of two for real single-molecule data (the 2006 STORM experiment went from 1.8 nm to about 4 nm). Estimate the practical localisation precision.
((c)) A researcher claims that two labelled sites 15 nm apart have been resolved using this single frame. Decide whether the claim is justified, using the sparsity and labelling requirements of localisation microscopy, and explain your reasoning in one or two sentences.