Cell culture
Murine motor neurons (NSC-34) were fixed in 4% paraformaldehyde (PFA), stained for F-actin using AF488-phalloidin using the manufacturer’s protocol (A12379; ThermoFisher Scientific, Eugene, OR) [18], and stained for nuclei using DAPI. Primary rat-derived Schwann cells isolated from neonatal Fisher rats [19] were fixed in 4% phosphate-buffered paraformaldehyde (PFA), transfected with lentiviral mCherry protein, and stained for nuclei using DAPI. Cells were plated on 18-mm #1.5 coverslips and mounted on glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) using refractive index-matching media (Mount Liquid Antifade, Abberior GmbH, Göttingen, Germany).
Animal tissue processing
All animal work was performed in accordance with institutional animal care and use committee protocols, and approved by the Massachusetts Eye and Ear Institutional Animal Care and Use Committee. Sciatic nerves from wild-type C57BL/6J mice were serially fixed in 2.5% glutaraldehyde and 2% osmium tetroxide solutions prior to resin embedding, ultramicrotome sectioning, and toluidine blue counterstaining for transmission electron microscopy (TEM) and light microscopy imaging as previously described [20]. Sciatic nerves were harvested from male and female adult Sox10-Venus (15–20 g) mice [21]. Sox10 is a transcription factor highly specific to oligodendrocytes and SCs of the peripheral nervous system. Sox10-Venus mice express high levels of Venus fluorescence in Schwann cell nuclei and cytosol. Nerves from Sox10-Venus mice were fixed by immersion in 2% PFA, followed by overnight cryoprotection in sucrose solution, cryosectioning at 1 µm for widefield microscopy, and stained with a myelin-specific fluorescent dyes (FluoroMyelin Red®, F34652 or FluoroMyelin Green®, F34651 Invitrogen, Carlsbad, Calif.) as previously described [22].
Human tissue processing
Written informed consent was obtained from patients with Massachusetts Eye and Ear Institutional Review Board approval and tissue processing methods performed in accordance with associated guidelines and regulations. Sural nerve autograft samples shuttling regenerating facial nerve motor axons across the face were obtained fresh from patients undergoing two-stage cross-facial smile reanimation [23]. Regenerating nerve specimens measuring roughly 3 mm in length were immediately fixed by immersion in 2% PFA, followed by overnight cryoprotection in sucrose solution, cryosectioning at 2 µm, and staining with a myelin-specific dye (FluoroMyelin Green®, F34651 Invitrogen, Carlsbad, CA).
Brightfield/Widefield Microscopy
Samples were imaged using an upright microscope (Axio Imager A.2; Carl Zeiss, Oberkochen, Germany), with a 40×/1.3 oil-immersion objective lens (EC Plan-Neofluar; Carl Zeiss), with transmitted light (brightfield color) and epifluorescence light (monochromatic) collected using cooled charge-coupled device cameras (Axiocam 503 color and monochrome; Carl Zeiss). Fluorescence was excited using a broadband light-emitting diode source (X-Cite 120 LED; Excelitas Technologies Corp., Waltham, MA), and a mercury lamp for brightfield illumination. Green fluorescence labelled samples were imaged using a GFP filter, while red fluorescence labelled samples were imaged using a TxRed filter. Images were acquired using ZEN 2Blue software (Carl Zeiss).
Electron Microscopy
Sections were imaged using a transmission electron microscope (FEI Morgagni 268, Eindhoven, Netherlands) and images captured with a digital CCD camera (2K, Advanced Microscopy Techniques, Woburn, MA).
SRRF Microscopy
For image acquisition, 100 frames acquired near the Nyquist sampling rate of the aforementioned widefield microscope were collected with a 100X oil-immersion objective lens (EC Plan-NEOFLUAR, Carl Zeiss, Jena, Germany) to generate individual SRRF frames. Images were acquired using a real-time SRRF-Stream enabled iXon Life 888 EMCCD camera (Andor Technology Ltd, Belfast, Northern Ireland), and aforementioned mono CCD camera using an open-source SRRF post-processing algorithm (NanoJ-SRRF [6]) in open-source image analysis software (ImageJ/Fiji [24]).
SIM Microscopy
Widefield and SIM images were acquired using the ONI Nanoimager system (Oxford NanoImaging, Oxford, UK) using a 100X 1.41NA oil-immersion objective lens (PlanApo, Olympus, Tokyo, Japan). Excitation lasers at 488 nm and 561 nm were used for fluorescence excitation, with signal collected using respective FITC and TRITC filters via a dual-color channel sCMOS camera with an exposure time of 30 ms per frame.
Confocal/STED Microscopy
Confocal and super-resolution images were obtained on a STED microscope (Leica SP8X STED, Leica Instrument) using a 592 nm depletion beam. Nerve sections from Sox10-Venus mice were excited using a 488 nm laser, and fluorescence collected with a hybrid detector using a spectral range of 500–550 nm. For STED microscopy, a HCX PL APO CS 100×1.4 numerical aperture oil objective (Leica Microsystems, Mannheim, Germany) was employed, with a scan speed of 400 Hz, 2048 pixels per line, time-gated detection of 1 ns, and 16-line averaging.
Resolution estimation
Resolution improvement was calculated using the Fourier ring correlation (FRC) [25]. The FRC approach estimates image resolution by transforming two independent images from spatial to frequency domain, and measuring the effective cutoff frequency.
Image segmentation
Myelinated axons were segmented and quantified from digitized images using commercial machine learning software (Aivia v8.5, DRVision Technologies LLC, Bellevue, WA) as previously described [9].