Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density

Abstract

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.

Recent applications of superresolution microscopy in neurobiology

“…An approach termed universal point-accumulation-for-imaging-in-nanoscale-topography (uPAINT) implemented by Giannone et al [25 ] used an anti-GluR2-ATTO647N antibody to map trajectories of AMPA receptors in cultured hippocampal neurons. These authors were able to map 189 trajectories of AMPA receptors in a single spine [25 ]. The combination of single-molecule subdiffraction imaging capabilities (i.e.…”

Advancing multiscale structural mapping of the brain through fluorescence imaging and analysis across length scales

“…Understanding how such genetic variation impacts synaptic molecular organization and signal transmission is therefore of central importance in resolving brain structure. Superresolution fluorescence imaging offers the ability to probe synaptic structure by imaging individual intact synapses with typically 20 nm spatial resolution, in principle across numerous molecular targets in a multiplexed manner [26,27]. Among several distinct super-resolution imaging approaches, localization microscopy enables the highest imaging resolution (approx.…”

“…A number of variants of PAINT have been developed since the seminal work of Sharanov and Hochstrasser. Universal PAINT (uPAINT) is one alternative super-resolution technique that employs binding and bleaching of high-affinity antibodies to achieve single-molecule imaging and has been used to map out the dynamics and organization of AMPA receptors on the post-synaptic membrane [26]. Other variants of PAINT have employed transiently binding nucleic acid probes that can be exchanged after imaging to enable multiplexed super-resolution microscopy [48], breaking the limit of four targets that can be imaged using conventional fluorescence microscopy due to spectral overlap.…”

Fluorescence nanoscopy. Methods and applications

“…Photoinduced activation may not be necessary: PALM with independently running acquisition (PALMIRA) exploits spontaneous activation and deactivation and asynchronous detection, which results in faster acquisitions [40]. When the interest is restricted to imaging the cell membrane, single particle tracking of photoswitchable fluorophores (sptPALM) and transiently labelling the surface with synthetic dyes that become fluorescent upon binding (point accumulation for imaging in nanoscale topography, PAINT) are two variations of stochastic LM that achieve higher number of localisations: sptPALM allows hundreds of individual molecules to be visualised and localised at the same time with tens of nanometres precision, mapping the trajectories of single molecules at high molecular density [41]; PAINT involves transiently labelling the surface of the cell such that a fluorescent signal appears as a diffraction-limited spot when the fluorophore binds to the membrane and disappears when it dissociates or bleaches [42][43][44]. PAINT and sptPALM are advantageous over conventional single particle tracking because many overlapping trajectories can be followed as long as the distance between fluorescent molecules at any time is greater than several times the width of their PSF.…”