Definition of single molecule localization microscopy

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Photonics Dictionary

single molecule localization microscopy: Single molecule localization microscopy (SMLM) is a group of super-resolution microscopy techniques that surpass the diffraction limit of traditional optical microscopes, allowing for the visualization of cellular structures at the nanoscale. Unlike conventional microscopy, which is limited by the diffraction of light, SMLM achieves super-resolution by precisely localizing individual fluorescent molecules within a sample.

The fundamental principle of SMLM involves the stochastic activation and precise localization of fluorophores (molecules that emit light) at different points in time. By acquiring the positions of individual molecules over multiple cycles, a high-resolution image of the sample can be reconstructed. Different SMLM techniques include stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and others.

Key steps in a typical SMLM experiment include:

Activation and imaging: Only a subset of fluorophores is activated and imaged at any given time. This allows for the precise localization of individual molecules.

Photobleaching or deactivation: After imaging, the activated fluorophores are often irreversibly photobleached or deactivated to prevent further emissions.

Iterative localization: The process of activation, imaging, and photobleaching is repeated multiple times, capturing different subsets of molecules in each cycle.

Data analysis: Computational algorithms are then applied to determine the precise locations of the individual fluorophores based on their emitted signals.

Image reconstruction: The final super-resolved image is reconstructed from the cumulative localization data, revealing details at resolutions far beyond the diffraction limit.

SMLM techniques have played a crucial role in advancing our understanding of cellular structures and dynamics, providing researchers with the ability to study biological samples with unprecedented detail. These techniques have been instrumental in elucidating the organization and interactions of biomolecules within cells.

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