Diffraction-unlimited all-optical imaging and writing with a photochromic GFP

Abstract

Lens-based optical microscopy failed to discern fluorescent features closer than 200 nm for decades, but the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off is making nanoscale imaging routine. Reported fluorescence nanoscopy variants switch these features either with intense beams at defined positions or randomly, molecule by molecule. Here we demonstrate an optical nanoscopy that records raw data images from living cells and tissues with low levels of light. This advance has been facilitated by the generation of reversibly switchable enhanced green fluorescent protein (rsEGFP), a fluorescent protein that can be reversibly photoswitched more than a thousand times. Distributions of functional rsEGFP-fusion proteins in living bacteria and mammalian cells are imaged at <40-nanometre resolution. Dendritic spines in living brain slices are super-resolved with about a million times lower light intensities than before. The reversible switching also enables all-optical writing of features with subdiffraction size and spacings, which can be used for data storage.

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

“…However, within the same color group, the difference in photon budget was less than twofold. In addition to the above-listed fluorophores, we also imaged rsFastLime (24) and rsEGFP (25). Both of these proteins gave relatively low photon budget (<60 photons per switching event), which would lead to relatively poor localization precision.…”

“…We thus did not further characterize these proteins. It is, however, worth noting that these proteins are excellent choices for a different mode of superresolution imaging [reversible saturable optical fluorescence transitions (RESOLFT)] due to the large number of switching cycles that they exhibit before photobleaching (24,25).…”

“…However, for mapping of protein spatial organization, a larger number of switching cycles per molecule would allow the target structure to be sampled more times, which is advantageous when it comes to time-lapsed imaging for probing dynamics. For a different superresolution imaging method, RESOLFT, reversibly switching fluorescent proteins with a large number of switching cycles (such as rsEGFP) is actually required (25).…”

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…Alexa 647, Dronpa, Dreinklang and rsEGFP) 3,[18][19][20] and (ii) irreversibly photoactivatable and photoconvertible (e.g. PA-GFP, PA-mCherry, mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2 and mMaple) [21][22][23][24][25][26][27] .…”

Three-dimensional deep sub-diffraction optical beam lithography with 9 nm feature size

“…However, the diffraction nature of light prevents us from achieving sub-diffraction or nanometre resolution in an OBL system 5 . Even for two-beam OBL based on the polymerization and photoinhibition strategy [6][7][8][9][10] , it has been impossible to realize the fabrication with feature size and resolution comparable to that achievable by EBL due to the lack of photoresins with large two-photon absorption cross-section, high mechanical strength and sufficient photoinhibition function.…”