Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes

Abstract

Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics (≈200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of ≈20–30 nm at acquisition times of 5–30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.

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

“…These probes can be activated in sparse numbers over time such that their images are optically resolvable and can be precisely localized 4 . These techniques have been exploited to image sub-cellular structures [5][6][7] , visualize protein (co)-organization [8][9][10] and quantify protein stoichiometry [11][12][13][14][15] .…”

Super-multiplex vibrational imaging

“…Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ , reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly 1–4 , it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm −1 ) and featureless nature of fluorescence spectra 5 that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis) 6–8 .…”

Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

“…[25][26][27][28] Fortunately, switching emitters to reach nanoscopical resolution largely maintains the versatility of the fluorescence readout. Consequentially, multicolor STED, 29 PALMIRA, 30 STORM, 31 and PALM 32 have recently been demonstrated. However, in all these cases, the separation of the signal was achieved by using a strategy known from conventional fluorescence microscopy: Markers with distinct activation or excitation spectra were imaged sequentially by choosing the appropriate illumination wavelengths, and the crosstalk between the different color channels was minimized by a careful selection of emission filters.…”