Wide-field subdiffraction imaging by accumulated binding of diffusing probes

Abstract

A method is introduced for subdiffraction imaging that accumulates points by collisional flux. It is based on targeting the surface of objects by fluorescent probes diffusing in the solution. Because the flux of probes at the object is essentially constant over long time periods, the examination of an almost unlimited number of individual probe molecules becomes possible. Each probe that hits the object and that becomes immobilized is located with high precision by replacing its point-spread function by a point at its centroid. Images of lipid bilayers, contours of these bilayers, and large unilamellar vesicles are shown. A spatial resolution of Ϸ25 nm is readily achieved. The ability of the method to effect rapid nanoscale imaging and spatial resolution below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven.diffusion controlled ͉ fluorescence ͉ single molecule F or the purpose of visualizing biological processes and structures, it is essential to develop new methods that can determine the spatial locations of molecules with the highest possible precision. The submolecular methods of spatial resolution such as x-ray and electron topography, atomic force microscopy, and near-field optical microscopy have contributed enormously toward this goal. However, new approaches that are not invasive to biological objects and are accessible to bulk, as well as surface structures, could have a significant impact and could address many new questions in the life sciences (1). Optical methods based on fluorescence detection do have single-molecule sensitivity and can be arranged to be nondestructive even when performed in natural environments, incorporating such complex and sensitive structures as living cells. In principle, optical responses can probe the entire 3D space. Although diffraction limits the spatial resolution of optical methods, many approaches have been developed recently that allow distance measurements on scales that are much shorter than the wavelength of the light.It is possible to specify the location of a single molecule with very high precision from measurements of its fluorescence by fitting the emitted intensity distribution to the 2D spatial parameters of the point-spread function (PSF). This approach has been shown to locate emitters with Ϸ1-nm precision (2). The distance between two optically different molecules can be estimated by FRET methods that can serve as molecular rulers at nanometer accuracy (3). Two molecules emitting light at different frequencies have also been resolved by means of PSF measurements. Even for molecules having the same spectra, the process of photobleaching causes the fluorescence source to be switched between molecules. When combined with a PSF measurement, this approach has been able to distinguish pairs (4) and quartets (5) of molecules with nanometer precision. Another approach has been to analyze the blinking trajectory of the emission of quantum dots to distinguish between a single dot and groups of them clustered on the nanom...

Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells

“…This enables us to quantify the oligomers formed during physiologically relevant aggregation reactions. Each image was acquired over 200 s; however, as PAINT‐based techniques are not limited by photobleaching,30 this time can be lengthened to localise a greater number of binding events in order to obtain a higher‐resolution image of the protein or the cellular structure of interest. …”

Super-resolution surface mapping using the trajectories of molecular probes

“…STORM 12 (stochastic optical reconstruction microscopy) and PALM 13 (photoactivated localization microscopy) are perhaps the best-known examples. PAINT 14 (point accumulation for imaging in nanoscale tomography) is a similar approach that has been applied at surfaces, using randomly adsorbed molecules from solution to identify surface features with super-resolution precision [14][15][16][17] . This is a powerful concept; however, the methods developed to date provide images that are based purely upon 'point counts' (a relative contrast mechanism) and lack the general ability to connect observed features with specific material properties and interactions.…”

“…This is a powerful concept; however, the methods developed to date provide images that are based purely upon 'point counts' (a relative contrast mechanism) and lack the general ability to connect observed features with specific material properties and interactions. In addition, these techniques are thus far limited to special cases of complementary DNA 15 on surfaces, local surface enhancement from hotspots 17 or where the surface selectively quenches fluorescence outside features of interest 14,16 . Our goal here is to develop a generally applicable super-resolution surface mapping method, where features observed in images can be directly related to specific surface functionalities.…”

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.…”