PALM reconstructs a spatial map by activating sparse subsets of labels, estimating each isolated fluorophore's position and combining many frames. Optical resolution depends on localization uncertainty, labeling density, drift and linkage; repeatedly observing one label cannot recover unlabeled structures.
A small subset of photoactivatable labels is fluorescent at any one time so that their overlapping point spread functions can usually be avoided. Repetition supplies many spatial localizations without requiring all labels to emit simultaneously.
Sparse photoactivation allows successive positions of individual labelled molecules to be linked into trajectories; activation of fresh subsets yields many tracks in living cells. This extends photoactivated localization microscopy from accumulated positions to mobility. For free two-dimensional diffusion with independent per-coordinate fluorescence localization precision , instantaneous frames give mean squared displacement . Motion blur, tracking errors, confinement and photobleaching must be accounted for before assigning mobility classes.[ref]
High-density mapping of single-molecule trajectories with photoactivated localization microscopy by Suliana Manley and others. Abstract and pages 155–156: combining sparse activation with single-particle trajectories
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Photoactivated localization microscopy (PALM) is a super-resolution imaging technique used in fluorescence microscopy to achieve spatial resolution beyond the diffraction limit of light. This technique is particularly important for visualizing cellular structures and protein interactions at the nanometer scale. Here's how it works: 1. **Photoactivation**: In PALM, a population of fluorescent molecules is initially in a dark or non-fluorescent state.