Supplementary Materialsijms-19-01150-s001. of individual images, the sample was illuminated with a 405 nm laser light for 10 ms. The dSTORM images were reconstructed from 15,000 frames. The localization events decided from Alexa Fluor 647 blinking are shown in red. Individual F-actin molecules bound by phalloidin conjugated to the commonly used cyanine dye Alexa Fluor 647 were localized with a 12 nm positional accuracy after drift correction for compensation of the mechanical sample movement. On average, we detected 3500 signals/m2 within a cell (= 3 cells). The blinking behavior of Alexa Fluor 488 was compared to Alexa Fluor 647: Fewer signals were detected for the SAG biological activity Alexa Fluor 488 labeled cells (740 signals/m2 as compared to 3500 signals/m2 for Alexa Fluor 647). However, the blinking characteristics are comparable: Alexa Fluor 488 showed on average 9 2 blinking events per 15,000 frames (SD = 0.1; = 45 analyzed image sequences) and Alexa Fluor 647 showed 10 3 blinking events (SD = 0.1; = 45 analyzed image sequences). In contrast to standard fluorescence microscopy, localization microscopy allows quantitative visualization of diffraction limited actin cytoskeleton substructures. The width of the peripheral actin network (i.e., the actin network at the edge of the fried-egg shape as seen in Physique 2c,f) varies from 140 nm to 565 SAG biological activity nm for Phalloidin SAG biological activity Alexa 488 and from 200 nm to 580 nm for Phalloidin Alexa 647, respectively. Similarly, the width of filopodia was decided: Physique 3a shows the image of the actin cytoskeleton recorded with diffraction limited fluorescence microscopy; whereas Physique 3d depicts the cytoskeleton from the same cell reconstructed by localization microscopy. In Amount 3b, the filopodium in the boxed area in 3a is normally depicted (furthermore the same filopodium at nanoscale quality, however, observed in Amount 3e in the boxed area in Amount 3d). The width of an individual filopodium (Amount 3b,e) was quantified by appropriate the combination section profile using a Gaussian function. Amount 3c displays the profile of the filopodium whose width was driven (in the diffraction limited picture a FWHM = 675 nm; for Phalloidin SAG biological activity Alexa 647). Open up in another window Amount 3 Evaluation of a typical fluorescence and a super-resolution picture of the actin cytoskeleton of the platelet using a spindle-like morphology tagged with Phalloidin Alexa 647 (a,d). Close-up of the spot of interest in the picture in (a,d) displays an individual filopodium (b,e). The strength profile from the filopodium cross section in the boxed region (b,e) continues to be fitted using a Gaussian function (c,f). In the suit a FWHM of 675 nm (c) or of 115 nm (f) continues to be driven for the filopodium width. Range club for (a,d) 3 m as well as for (b,e) 1 m. The IFITM2 width from the filopodium was driven in the reconstructed dSTORM picture (Amount 3f): A FWHM of 115 9 nm was assessed for Phalloidin Alexa 647 and 106 3 nm for Phalloidin Alexa 488, respectively. From the utilized label Irrespective, the same morphological features (width of filopodia and peripheral actin network) at the same quality (12 nm for both fluorophores) have already been driven. 3. Conclusions and Debate In conclusion, we showed our optimized process allows for recording qualitatively comparable images with Alexa Fluor 488 (compared to popular Alexa Fluor 647) labeled actin filaments. We showed for the first time the actin cytoskeleton of three different platelet morphological claims resolved at a resolution beyond the diffraction limit. In contrast to the dSTORM images, the conventional fluorescence microscopy images of filopodia appeared as diffraction limited constructions:.