N achroplan

Cells were seeded on rat-tail collagen I coated plates for 24 h before indicated treatments. Mitochondria and nuclei were labeled with MitoTracker Green (100 nM) and Hoechst 33342 (20 μM) for 30 min at 37 °C, respectively. Phenol red free media supplemented with 10% FBS and 2 mM l-glutamine and antibiotics was used for all imaging performed on a Zeiss Imager.Z1 equipped with a N-Achroplan 40 × /0.75 water immersion lens and an AxioCAM MRm digital camera; images were captured using AxioVision 4.8 and Zeiss Zen software. At least 300 cells per condition were quantified. The Z-stack images were processed using Image J software (NIH) and the mitochondrial length was measured using NIS Elements software (Nikon, USA). Scale bars = 25 μm.

Micro-Raman spectra were obtained with an Alpha 500R confocal Raman microspectroscopy system (WITec GmbH, Germany) coupled with a helium-neon (He-Ne) continuous 633 nm laser beam (35 mW @ 633 nm, Research Electro-optics, Inc., USA). The excitation laser beam was collimated into a 20× objective lens (NA = 0.85, N-Achroplan, Zeiss, Germany) for Raman excitation. Raman photons were collected by the same objective lens and transmitted through a holographic edge filter to a multi-mode optical fiber (50 μm diameter) to the spectrometer (UHTS300, WITec GmbH, Germany), which was equipped with a resolution about 3 cm⁻¹ over a spectrum range of 0–2400 cm⁻¹. The spectra were recorded using a back-illuminated, deep depletion CCD camera containing 600 × 200 pixels (Du401A-BR-DD-352, Andor Technology, UK) working at −60 °C. The spectral data were acquired point-by-point over each kind of skin lesion with 3 s integration time. Before the experiment, a standard tungsten lamp (RS-3, EG&G Gamma Scientific, USA) was used for calibrating the spectral response of the system, and the Raman spectrum of silicon (520 cm⁻¹) was measured to calibrate the wavelength position.

For ex vivo calcium imaging experiments, 5–7 days old naïve males were briefly anesthetized on ice and brains were dissected out in calcium free external saline (ES) containing: 103 mM NaCl, 3 mM KCl, 5 mM TES (N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid, a buffer chemical with peak performance around pH7.5), 10 mM trehalose, 10 mM glucose, 26 mM NaHCO₃, 1 mM NaH₂PO₄, 4 mM MgCl₂, 7 mM sucrose, pH 7.4, 275 mOsm (Gu and O'Dowd, 2006 (link)). The brain explants were transferred into a custom-made imaging chamber and mounted with anterior side up. Brains were perfused with ES supplemented freshly with 2 mM calcium, at speed 2 mL/min, pre-saturated with mixture of 95% O₂/5% CO₂. All two-photon imaging were performed using 40x N.A. 0.75 water-immersion objective (N-Achroplan, Zeiss), on LSM 7 MP microscope (Zeiss) with a Ti:sapphire laser (Chameleon Vision II, Coherent, Santa Clara, CA). GCaMP was excited at 900 or 920 nm and emission signals were collected by GaAsP photomultiplier tubes (PMTs). Frame images (256 × 256 pixels) were acquired at 5–10 Hz. The region of interest (ROI) covers the entire bilateral medial γ5 lobe in MB. For consistency, imaging focus was kept approximately at the same level in different animal guided by axon position of M6 or aSP13.

Dissected spines were fixed in 4% PFA in PBS for 48 hr, decalcified in 20% EDTA and embedded in paraffin. Spines used for calcified sections were fixed for 2h, treated with 30% sucrose, OCT embedded and snap‐frozen. 7 µm mid‐coronal sections were cut from 6 caudal levels (Ca3‐9) of each mouse and stained with Safranin‐O/Fast Green/haematoxylin for assessing histology or Picro‐Sirius Red, to visualize the collagen content. Alizarin Red staining was used to detect calcium. Staining was visualized using a Axio Imager 2 microscope (Carl Zeiss) using 5×/0.15 N‐Achroplan or 20×/0.5 EC Plan‐Neofluar objectives (Carl Zeiss) or a polarizing microscope (Eclipse LV100 POL, Nikon) using 10×/0.25 Pol/WD 7.0 objective, Digital Sight DS‐Fi2 camera, and NIS Elements Viewer software. To evaluate degeneration, mid‐coronal sections from >4 caudal discs per mouse were scored using a modified Thompson Grading scale by 4 blinded observers (Choi et al., 2018).

We constructed an optical configuration for the back-scattering RS system (Figure S1 in the Supporting Information) as described previously [37 (link),38 (link)]. In brief, a single continuous wave laser of 532 nm (Excelsior-532-200, Spectra-Physics, Santa Clara, CA, USA) with working power of 40 mW was coupled into an inverted microscope (Axiovert 200, Zeiss, Oberkochen, Germany). A 100 ×/1.25 oil objective (N-Achroplan, Zeiss, Oberkochen, Germany) with a working distance of 0.29 mm was used for RS measurement. Overall, considering the signal-to-noise ratios (SNR) of RS and photodamage to the cell, an optimal laser power of 20 mW on the cancer cell was used. The back-scattering Raman light was collected with the same objective and was directed into a spectrometer (SpectraPro2300i, 1200 g/mm blazed at 500 nm, Acton Research Co., Acton, MA, USA). A pinhole of 100 μm diameter was used to create a confocal system. A notch filter (532 nm, Thorlabs, Newton, NJ, USA) was placed before the entrance of the spectrometer to filter Rayleigh scattered light from the sample. Each RS was recorded with a high sensitivity liquid nitrogen-cooled spectroscopic CCD (Spec-10:400BR/LN, Princeton Instruments, Trenton, NJ, USA).