via: Leica Science Lab
Imaging living cells is always a challenge for most of the common super-resolution principles. Unlike the STORM and PALM methods, universal Point Accumulation for Imaging in Nanoscale Topography, or uPAINT offers super-resolved images and single molecule trajectories at very high densities with the Leica SR GSD 3D
In order for these approaches to work, the vast majority of the molecules within the field of view must be in a non-emitting state. Stochastic Optical Reconstruction Microscopy, or STORM places most of the population in a dark state with high power laser illumination, and the specimen has to be embedded in oxidizing or reducing buffers to stabilize the off state of the fluorophore.
Photo Activated Localization Microscopy, or PALM uses photo-activated/convertible fluorescent proteins that can be “turned” on and off. The major challenge is achieving super resolved images of native proteins on living cells. That’s where uPAINT comes in.
uPAINT is a technique to circumvent these issues by dynamically imaging single-molecule tracks on the cell surface under oblique illumination. The resulting image is a super resolved tracking of native biomolecule behavior at the surface of a living cell.
Contrary to traditional localization microscopy methods utilizing stochastic fluorescent emission for imaging a small subset of molecules, uPAINT creates a temporal separation of fluorescence signals by imaging low concentrations of fluorescent ligands as they bind to and interact with their targets inside a small light volume. The uPAINT method achieves temporal separation of fluorescence signals by imaging low concentrations of fluorescent ligands as they bind to and interact with their targets inside a small light volume.
Unlike the relatively short and insertion dependent fluorescence of lipid probes used for PAINT, the signals from ligands used for uPAINT is limited by photobleaching of the dye rather than ligand dissociation rates. uPAINT circumvents the usage of harmful buffers or genetically modified proteins and accordingly enables observation under near natural conditions.
uPAINT experiments require periods of ten seconds or more to reveal the movement and organization for your proteins of interest. In those circumstances, drift can be corrected by image to image detection of a bead’s position.
After acquisition, the Leica SR GSD 3D software will automatically reposition each image by the bead’s localization. This drift correction mode is essential when studying nanoscale clusters for example.
Static as well as dynamic data of proteins can be gathered with one microscope enabling super-resolved examination of the organization plus the dynamics of a protein at endogenous levels on a living cell.
For more information check out all the Science Lab articles on the SR GSD 3D