Tcs sp8 gsted

4 × 10⁵ RNVCs were plated overnight on 35 mm culture dishes coated with 10 µg/mL laminin, and 24 h later, cells were treated for 6 h with 1 µM or 10 µM of different compounds. Cells were incubated with Mitotracker Red 580 at 200 nM for 20 min at 37 °C, then with 4 μM calcein (Life Technologies, Carlsbad, CA, USA) for 10 min at 37 °C. Z stack images were acquired with a Leica (TCS SP8 gSTED) inverted confocal laser scanning microscope (Mannheim, Germany) equipped with a WLL Laser (495 nm excitation wavelength for calcein and 580 nm for Mitotracker Red 580). Green fluorescence emission was detected with 505–550 nm wide emission slits and 585–700 nm wide emission slits for the red signal under a sequential mode. The pinhole was set at 1.0 Airy unit, and 12-bit numerical images were done with the Leica Application Suite X software (Version 3.5.5; Leica, Wetzlar, Germany).
Mitochondrial network and cell volume 3D model were reconstructed by using the IMARIS software 9.7 version (Bitplane Company, Zurich, Switzerland); consequently, cell volume, mitochondria number, and volume were analyzed using the volume and surface rendering processes.

Indirect immunofluorescence was performed after paraformaldehyde fixation and permeabilization with 0.1% Triton X-100 or 0.5% saponin. For imaging of transferrin endocytosis, the living cells were first serum-starved for 30 min, followed by exchange for medium supplemented with Alexa Fluor-conjugated transferrin (Invitrogen) for the indicated times. For TIRF microscopy, HEK293 cells were cultured and transfected using CALM-GFP and AP2 μ2-mCherry as detailed previously (Gage et al., 2005; Taylor et al., 2011 ). Cells were plated on clean coverslips on the same day as transfection and imaged 48 hr later to allow several cell divisions and to give transfected cells time to adapt moderate protein overexpression (Taylor et al., 2011, 2012 ). To determine CCP maturation time in CALM knockdown cells, HT1080 cells were transfected with either CALM or control siRNA followed by transfection with CALM-GFP and AP2 μ2-mCherry. The maturation time of CCPs was determined as described previously. For STED super resolution microscopy, samples were fixed and processed as described above. For microscopy, a Leica TCS SP8 gSTED equipped with a 100×/1.4 Oil STED Orange lens and a 592 nm depletion laser was used. Further details on STED imaging and quantification are summarized in the Supplemental Experimental Procedures.

We obtained PL images by a fluorescence microscope (Leica TCS SP8 gSTED) with excitation wavelength of 488 nm at room temperature. Raman and PL spectra were measured by using a confocal-microscope based Raman microscopy (Jobin Yvon LabRAM HR-800) with 488 nm excitation (CW laser, COHERENT Sapphire 488 LP). In measurements of Raman spectra, a notch filter was used to filter out the intense signal from Rayleigh scattering. An objective lens (100 x, 0.9 NA) was used to focus the laser light onto a sample and collect the backscattered light from the sample. Raman and PL signal were detected with a charge-coupled device. In temperature dependence measurements, we placed WS₂/hBN in a cryostat (CryoVac KONTI-Cryostat-Micro) with continuous flowing of liquid N₂ under vacuum of ~10⁻⁶ Torr. Temperature was controlled by a CryoVac TIC 304-MA.