Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores

Abstract

We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

“…S7). The z coordinates of the molecules can be obtained by a variety of means for 3D super-resolution imaging (21)(22)(23)(24)(25)(26), and indeed 3D STORM has been demonstrated in live cells (31). However, the use of diffusing membrane probes presents an additional challenge, especially when the shapes of the single-molecule images are used to determine their z-positions.…”

“…It has been further demonstrated that many conventional dyes can be used for super-resolution imaging based on photoswitching/bleaching and localization of single molecules (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Using single-molecule-based super-resolution methods, 3D resolutions down to approximately 10 nm have been demonstrated for fixed samples (21)(22)(23)(24)(25)(26), and live-cell imaging has also been achieved with spatial resolutions of 20-60 nm at time resolutions ranging from 0.5 s to 1 min (27)(28)(29)(30)(31)(32).…”

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

“…These methods can achieve precisions below 100 nm in 3D over a 2-μm range (14,19,20). In addition, sub-10-nm precision can be obtained via interferometry (21); however, these methods severely restrict the sample geometry, have a shallower operational depth of field (∼650 nm) (22), and are not yet conducive to live-cell imaging.…”

Fluorescence nanoscopy. Methods and applications

“…It is worth noting that localisation precision is not equivalent to resolution: The resolution achieved with LM strategies depends very much on the available photons, but also on the labelling density and the experimental configurations. For biological samples, it is generally around 20 nm in the lateral dimension (up to ≈4 nm [32]) and around 70 nm in the axial dimension (up to ≈6 nm [33]). …”

“…Superb lateral and axial resolution (≈4 and 5.4 nm, respectively [32,33]) has been achieved sandwiching the sample between two objectives to exploit the self-interference of emitted photons while taking advantage of the extra budget of photons available for improved localisation in all directions (interferometric PALM, iPALM or 4Pi-LM) [32,33,61]. This approach is limited to a 1-μm-thick optical layer (although the sample can be thicker) and is constrained by the severe complexity of the experimental setup.…”