Computational protocol: Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

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[…] ILSEM was performed on the same day as sectioning using a SECOM light microscope platform (Delmic B.V., Delft) with Nikon Plan Apo 100×/1.4 objective and vacuum compatible immersion oil (Delmic B.V., Delft), mounted on a Quanta 250 FEG SEM (FEI Company, Eindhoven). A schematic illustrating the overall imaging workflow adopted for sequential fluorescence, super-resolution, and SEM imaging is shown in .Diffraction-limited imaging was performed as previously described (). Briefly, YFP or GFP fluorescence was stimulated by excitation with a 488 nm laser light source and multi-band filters (Di01-R405/488/594 dichroic, FF01-446/532/646-25 emission; Semrock, Rochester NY). Individual fluorescence images were collected using an EMCCD camera (iXon 897 Ultra; Andor Technology, Belfast) with exposure time of 2 s, and power density of 0.5 W/cm2 at the sample level. During all fluorescence imaging phases, the chamber was maintained at a partial pressure of 200 Pa, created using water vapour.For super-resolution localisation-based imaging of specimens containing multiple fluorophores using high laser powers, it was necessary to further filter emission wavelengths using an individual bandpass filter (FF01-520/35-25; Semrock, Rochester, NY), which was placed directly in front of the camera. YFP or GFP fluorescence was again stimulated by excitation with a 488 nm laser light source at a power density of 330 W/cm2. Control of lasers and EMCCD camera were transferred to Micromanager () and the following camera parameters were set: −85 °C temperature set point, photon count convert, frame transfer on, EMCCD gain 200, preamplifier gain +3, readout speed 17 MHz, vertical clock speed +1.70. Sequences of ∼30,000 images were collected, each with an exposure time of 40 ms. The image sequences were processed for analysis as detailed below using ThunderSTORM ().For SEM imaging, the system was pumped to high vacuum (∼10−3 Pa). The vCD backscatter detector (FEI Company, Eindhoven) was used at a working distance of 7.3 mm, and inverted contrast images were acquired (2.5 keV, spot size 3.5, 30 μm aperture, and pixel dwell time of 60 μs for a 3,072 * 1,536 image frame). [...] The theoretical resolution of the SECOM platform using a 488 nm light source and 100×/1.4 objective lens was calculated as 213 nm. The full width half-maximum (FWHM) of the system was measured using 30 nm fluorescent beads at ∼265 nm. The calculated pixel size of the iXon EMCCD camera was 107.8 nm.Several freely available reconstruction packages were tested during preliminary super-resolution data acquisition in order to determine which worked better with the data generated by the ILSEM-based system, including 3B (), CSSTORM (), Localizer (), Octane (), QuickPALM (), RainSTORM () and RapidSTORM (). The patterns of particle localisation obtained were remarkably similar, but with tremendous variation in processing time and flexibility of operation from a user standpoint (refer also to for a review). The Fiji plugin ThunderSTORM was used to reconstruct all data from the imaging sequences shown here (, ). ThunderSTORM represented an acceptable balance between overall processing time and perceived quality of output when compared to the electron imaging ‘ground truth’ data; it also offered more flexibility for interactive data post-processing.Camera setup was performed according to the EMCCD specification and EM gain applied during acquisition. Given the calculated system resolution, a fitting radius of 4 pixels was used, with sigma of 1.6 pixels, and multi-emitter fitting analysis. Post-processing of the detected molecule localisations was carried out in the following sequence: remove duplicates (uncertainty_xy); filter (intensity > 5 & intensity < 2,000 & frame > 500); density filter (radius 100 nm, neighbours 5); drift correct (cross correlation, bins 5, magnification 5.0); merging (xy distance 20, off frames 1).Reconstructions were generated using averaged shifted histograms at a magnification of 10× (for low magnification overlays; histogram mapped to a range of 0–30) or 20× (for high magnification overlays; histogram mapped to a range of 0–14.74) to avoid interpolation when overlaying onto electron micrographs. A ‘Fire’ LUT was applied before exporting the images as 16 bit RGB TIFFs for overlay onto the corresponding electron micrographs.Electron micrographs were adjusted to reduce image noise and enhance contrast, and composite images of dual signals were generated as described previously (). Movies of the image sequences were generated using iMovie (Apple). As the original image sequences were recorded with 16 bit greyscale depth, were processed in Fiji (contrast normalisation; 0.1% saturated pixels) for on-screen presentation. […]

Pipeline specifications

Software tools ThunderSTORM, QuickPALM, rainSTORM, rapidSTORM
Application Super-resolution imaging
Diseases HIV Infections