Bx51wi microscope

LV myocytes fixed in paraformaldehyde were stained with the nuclear dye Hoechst (10 μM, Sigma). Images were collected with a fluorescent inverted microscope (Olympus IX71) equipped with a CCD camera (Hamamatsu ORCA-R2). Cell length and area were evaluated using ImageJ software. Myocyte volume was then computed assuming an elliptical cross-section, in which the major axis corresponds to the average cell width, and the minor axis was computed after establishing the ratio between the major and minor axis, using three-dimensional optical section reconstruction by two-photon microscopy (BX51WI Olympus microscope coupled with a Bio-Rad Radiance 2100MP system) and image analysis (ImageJ). Three-dimensional reconstructions were obtained using second-harmonic generation signal of sarcomeric structures65 (link).

NPCs were terminally differentiated on Matrigel coverslips and aligned and unaligned two-dimensional nanofiber mats to determine impact under long-term growth conditions. Plasma treated polycaprolactone (PCL) nanofiber mats were obtained from Nanofiber Solutions. Fiber mats were coated with Matrigel and seeded at a density of 2 x 10⁴ in TD media with media changes every 3 days. To visualize neurite alignment and assess phenotype, preparations were immunostained with TUJ1 primary antibody as described below. Epifluorescence images were obtained with a BX51WI Olympus microscope with Orca Flash4.0 V2 Digital CMOS camera. Images were analyzed by fast Fourier transform (FFT) as described elsewhere [53 (link)], averaging intensities in a radial band 20–40 μm from the image origin and plotting against corresponding angle from the origin in 1° increments. From this plot, the full width-half maximum (FWHM) was calculated as a measure of strength of alignment.

Ca²⁺ imaging was performed according to a previously published protocol^{69 (link)}. Briefly, cells were loaded with 5 mM of fluo-4 fluorescent Ca²⁺ indicator (Molecular Probes) in the presence of Pluronic F-127 (Molecular Probes) to allow the recording of intracellular Ca²⁺-transients. For pacing, scaffolds were plated on a 35-mm optical plate (Matek) with field stimulation electrodes (RC-37FS; Warner Instruments) and paced using a stimulus isolation unit (SIU-102; Warner Instruments), by applying 5 ms-suprathreshold bipolar stimulation pulses up to 50 mA. Intracellular Ca²⁺-transients were recorded using a Zeiss laser-scanning confocal imaging system (Fluo-view; Olympus) mounted on an upright BX51WI Olympus microscope equipped with a X60 water objective. Data were analyzed utilizing a MatLab-based custom-written software.

HEK293T cells (human embryonic kidney 293T cells, ATCC, USA) were tested for mycoplasma and culture in DMEM (Gibco, USA), containing 10% fetal bovine serum (Gibco), penicillin (100 U ml⁻¹, Gibco) and streptomycin (100 μg ml⁻¹, Gibco). They were transfected using the Effectene Transfection Reagent (Qiagen, Germany) containing 400 ng of mANO1-IRES-GFP or mANO2-EGFP-N1 for 16 h. On the day of the experiment, the transfected cells were trypsinized and transferred on poly-D-lysine-coated coverslips in a 12-well plate. The coverslip with attached cells was placed on the stage of a BX51WI Olympus microscope (Japan) and perfused with HEPES-based solution. The extracellular solution was composed of HEPES, 10 mM; NaCl, 150 mM; KCl, 3.2 mM; CaCl₂, 2 mM; MgCl₂, 2 mM; glucose, 5.6 mM; and sucrose, 22 mM (pH adjusted to 7.2 with NaOH). Visually guided whole-cell patch recordings were obtained from GFP-positive HEK293T cells using borosilicate patch pipettes filled with high Ca²⁺ internal solution CsCl, 146 mM; Ca-EGTA-NMDG, 5 mM; MgCl₂, 2 mM; HEPES, 8 mM; sucrose, 10 mM; Na-ATP, 4 mM; and Na-GTP, 0.3 mM (pH adjusted to 7.3 with CsOH). Data were collected with a Multiclamp 700B amplifier (Molecular Devices) using the Clampex 9 acquisition software and digitized with Digidata 1322A (Molecular Devices).

The sample is placed under a specially prepared BX51 WI Olympus microscope coupled to an InGaAs camera cooled at −10 °C (model Xeva 1M with 320 × 256 pixels from Xenics) (Figs 2b,d and 4d,e). The images of Figs 2b,d and 4d,e are recorded with a × 10 infrared objective with numerical aperture (NA)=0.25, also from Olympus. To obtain the images of Fig. 4d,e, a Glan–Thomson polarizer is inserted inside the microscope and successively aligned along the vertically oriented and horizontally oriented nanorods.