Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets

Abstract

Fluorescence nanoscopy provides means to discern the finer details of protein localization and interaction in cells by offering an order of magnitude higher resolution than conventional optical imaging techniques. However, these super resolution techniques put higher demands on the optical system and the fluorescent probes, making multicolor fluorescence nanoscopy a challenging task. Here we present a new and simple procedure, which exploits the photostability and excitation spectra of dyes to increase the number of simultaneous recordable targets in STED nanoscopy. We use this procedure to demonstrate four-color STED imaging of platelets with e40 nm resolution and low crosstalk. Platelets can selectively store, sequester, and release a multitude of different proteins, in a manner specific for different physiological and disease states. By applying multicolor nanoscopy to study platelets, we can achieve spatial mapping of the protein organization with a high resolution for multiple proteins at the same time and in the same cell. This provides a means to identify specific platelet activation states for diagnostic purposes and to understand the underlying protein storage and release mechanisms. We studied the organization of the pro-and antiangiogenic proteins VEGF and PF-4, together with fibrinogen and filamentous actin, and found distinct features in their respective protein localization. Further, colocalization analysis revealed only minor overlap between the proteins VEGF and PF-4 indicating that they have separate storage and release mechanisms, corresponding well with their opposite roles as pro-and antiangiogenic proteins, respectively.

Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force‐dependence

“…To allow clear identification of FAs, we analyzed their spatial localization along filamentous (F‐) actin at 40‐nm resolution by three‐color STED microscopy, utilizing a recently demonstrated imaging approach that exploits differences in photo‐stability between different fluorophores . As expected, we observed enrichment of FA proteins at the ends of stress fibers, which identified these areas as FAs (Figs and ).…”

Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection

“…This clearly demonstrates that the choice of markers in relation to the detection windows plays a crucial role. The number of distinguishable markers could be further increased by combining the present hyperSTED setup with other means of discrimination, for example excitation multiplexing21, fluorescence lifetime imaging2223 or photostability18, as previously reported. This is feasible without changes to the hardware, keeping the setup simple and stable.…”

“…To our knowledge, no live-cell super-resolution experiments with three or more separated species have been reported. Even when considering fixed cellular specimens, only a handful of reports were found involving four or more super-resolved fluorescent markers, realizing the nanoscopy methods called GSDIM15, STORM1617, STED18 and PAINT19. The respective experiments used elaborate imaging schemes and exploited a combination of excitation, emission, lifetime and/or photostability to obtain the separation of the various fluorophores.…”

Phase angle encoded upconversion luminescent nanocrystals for multiplexing applications

“…Even so, it has always been of high interest to develop new encoding dimensions to meet the increasing requirements of various applications. For instance, for implementing stimulated emission depletion (STED) microscopy (where a normal Gaussian excitation beam and a doughnut shaped emission depletion beam are used coaxially to achieve high spatial resolution 14 ) in multi-channels, non-spectral multiplexing strategies are desired due to the challenge of aligning multiple beams 15 , and this demand is not yet met.…”