Single Molecule Localization Microscopy

Understanding molecular dynamics and/or proteins nano-organization requires high spatial and temporal resolution. However, in conventional light microscopy, spatial resolution is limited by the diffraction of light through the optical system, and reaches a lateral resolution around 250 nm. Super-resolution microscopy and in particular Single Molecule Localization Microscopy techniques (SMLM) allow to overcome this barrier to get a lateral resolution around 20 nm.

At the BIC, we offer training and expertise on sample preparation, image acquisition, image post-processing and analysis for PALM, spt-PALM, u-PAINT, DNA-PAINT and dSTORM

Contact: Magali Mondin

 

 

When fluorescent emitters are isolated, their precise localization can be calculated from the PSF fitting. SMLM techniques consist in manipulating the samples so that individual fluorescent molecules emits photons  separated in space and time.

 

 

 

 

 

 

 

The acquisition is a temporal sequence where on each image a few molecules are detected, located and all localizations are then accumulated on the super-resolved reconstruction.

 

 

 

 

 

In this example, the left image represent the low resolution bulk fluorescence acquisition, the right image is the reconstructed image after localization of individual molecules over the total acquisition.

 

 

 

To be able to collect a maximum number of photons per individual emitter, SMLM techniques are restricted to a small volume of illumination and detection. They are thus mainly used in cell culture.

There are 3 main families of SMLM techniques that use different strategies to label the sample to get single molecule detections:

• PALM / spt PALM mainly use protein fusion with photo-convertible or photo-activatable fluorescent proteins like mEOS. PALM is compatible with live (tracking) and fixed samples (localization)
• dSTORM relies on the labelling of endogenous proteins through an immunolabeling. The blinking of the sample is obtained using intrinsic photswitching of fluorescent dyes when placed in a specific environment. dSTORM is particularly adpated to fixed samples (localization)
• DNA-PAINT uses the complementarity in between 2 single-strand DNA sequences, one linked to the antibody or the probe recongnizing the target, the second one linked to a fluorophore is diffusing in the observation media.. The blinking is obtained by controlling with environmental conditions the binding/unbinding frequency between the 2 DNA strands. DNA-PAINT is particularly adpated to fixed samples