Newton du970n bv 353

Magnesium fluoride (MgF 2 ) slides were pre-treated with BD Cell-Tak™ Cell and Tissue Adhesive in a 48-well tissue culture plate (BD Biosciences, 5.2 µL in 300 µL of sodium bicarbonate buffer, pH = 8 for 40 minutes at room temperature). Following two washing steps with ddH 2 O, MgF 2 slides were incubated with the cell suspension in PBS for 30 minutes. Slides were then fixed with 4% v/v PFA and washed gently with PBS before analysis. Raman spectral imaging was performed on a confocal Raman microscope (alpha300R+, WITec, Ulm, Germany). A 532 nm laser (Compass Sapphire, Coherent, Gottingen, Germany) was used for excitation. The laser beam was focused through a 63×/1.0 NA water immersion microscope objective lens (W Plan-Apochromat, Zeiss, Oberkochen, Germany). The Raman spectroscopy signals were dispersed by a grating (600 g mm -1 ) spectrograph (UHTS 300, WITec, Ulm, Germany) and the spectra acquired with a thermoelectrically cooled CCD detector (Newton DU970N-BV-353, Andor, Belfast, UK) with a spectral resolution of 3 cm -1 . Raman spectroscopy images ∼150 × 150 μm were produced with 400 nm spatial resolution, 1 s integration time and one accumulation per pixel. Spectral collection was centered at 2000 cm -1 (0-3000 cm -1 ). The Control FOUR software (version 4.0, WITec) was used for measurement and Project FOUR Plus (version 4.0, WITec) for spectral data processing.