Tof sims 5

ToF-SIMS imaging was performed with a TOF-SIMS V (IONTOF GmbH, Münster, Germany), with a bismuth liquid metal ion gun as a primary ion source and a C₆₀ 10-keV ion source as a sputter source. Mass spectra in positive ion mode were recorded by using Bi₃⁺ primary ions at 25 keV with a pulsed primary ion current of 0.25 pA. Delayed extraction mode was employed to obtain images with high spatial (at best 400 nm) and high respective mass resolution (approx. 5000 at m/z 300). Multiple images from each section were recorded in areas ranging from approx. 120 µm × 120 µm–450 μm × 450 µm on the skin sections using a raster of 256 × 256-pixels.

The Raman spectra were acquired on an inVia-Reflex (Renishaw) at an excitation wavelength of 532 nm and ATR-FTIR (attenuated total-reflection Fourier transform infrared) spectra were obtained on an Excalibur 3100 (Varian). The elemental chemical composition and chemical structure were determined by X-ray photoelectron spectroscopy (XPS, PHI QUANTERA-II equipped with a monochromatic Al K_α source). The analyzer was operated at a pass energy (Ep) of 280 eV for wide scans and 26 eV for fine scans leading to an instrumental resolution of 1.00 eV for wide scans and 0.025 eV for fine scans. The data were collected at a take-off angle of 45° and data analysis and multi-peak fitting were performed by the Multipak software. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was performed on the TOF-SIMS V (ION TOF GmbH) with 30 keV Bi_n⁺ as the primary ion source. The negative spectra were obtained from 500 × 500 μm² areas by focusing the Bi⁺ primary ions (less than 0.01 pA of pulsed current) in the “burst alignment” mode at a 10 kHz pulsing rate and 120–130 ns pulse width. The surface potential was measured by a Kelvin probe force microscopy with amplitude modulation (KPFM-AM, Multimode 8, Buker) in the tapping mode on a Multi75E-G (budget sensors) probe in air at room temperature.

Cells for TOF-SIMS analysis were harvested and prepared in the same manner as cells examined with the NanoSAM, except they were not coated with gold and they were mounted on copper stubs with double-sided tape. TOF-SIMS mass spectra and ion maps were obtained with a secondary ion mass spectrometer TOF-SIMS V (ION-TOF GmbH, Münster, Germany) fitted with a high mass resolution (>10 000) time-of-flight analyzer. Secondary ion mass spectra were recorded from clumps of cells in an approximately 20 μm × 20 μm area of the sample. During measurement the analyzed area was irradiated with pulses of 30 keV Bi^{1 +} ions from a primary ion gun at a 10 kHz repetition rate and a flood gun (low-energy electrons) was used to compensate for any surface charging. A 2.5 kV argon cluster gun was also used to sputter the samples in a 100 μm x 100 μm region to allow for depth profiling analysis. Mass spectra were recorded from m/z 0–200 and intensities of the negative ions C^-, NH^-, O^-, OH^-, P^-, and S^- were monitored. Intensities were normalized based on total intensity at each sputtering time to allow for comparison between samples, to account for any operational variability between runs [15 ] and to negate any difference in the amount of cell material between samples.

Mandibular bone specimens after micro‐CT analysis (N = 3 each) were dehydrated in an ascending series of alcohol and xylene without decalcification and then embedded with the Osteoresin embedding kit (Wako Pure Chemical Industries, Osaka, Japan). Sagittal sections of the embedded specimens were mirror‐polished with waterproof abrasive paper. Time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS; TOF‐SIMS V, ION‐TOF GmbH, Munster, Germany) was performed using a spectrometer equipped with a pulsed Bi+ liquid ion gun operated at 25 kV. The system vacuum was held below 10⁻⁶ Pa throughout the measurements. The average primary ion current was 1.25 pA, and the images were acquired in an area of 500 × 500 μm.

ToF-SIMS analyses were conducted using a TOF-SIMS V (ION TOF, Inc. Chestnut Ridge, NY) instrument equipped with a Bi_n^m+ (n = 1–5, m =  1, 2) liquid metal ion gun, Cs⁺ sputtering gun and electron flood gun for charge compensation. Both the Bi and Cs ion columns are oriented at 45° with respect to the sample surface normal. The instrument vacuum system consists of a load lock for rapid sample loading and an analysis chamber, separated by the gate valve. The analysis chamber pressure was maintained below 5.0 × 10⁻⁹ mbar. For depth profiles acquired in this study, 3 keV Cs⁺ with 18 nA current was used to create a 120 µm × 120 µm area, and the middle 30 µm × 30 µm area was analyzed using 0.4 pA Bi³⁺ primary ion beam. The analysis was done with the non-interlaced mode with three cycles of analysis and one cycle of sputtering to improve the detection limit. The positive secondary ion mass spectra were calibrated using NH₄⁺, Pb⁺, PbI⁺, CsPb⁺, and CsPbI⁺.

After the BMSCs were cultured on Blank, PE, Par, and PArN for 7 days, the samples were rinsed with PBS twice, fixed in 4.0% paraformaldehyde overnight, and washed again with PBS twice. They were dehydrated in a series of ethanol and tert-butanol and freeze-dried for 24 h. SIMS was conducted on a time-of-flight secondary ion mass spectrometer (TOF-SIMS V from ION-TOF GmbH, Munster, Germany) and a Bi₁⁺ liquid metal ion gun at 30 keV and 45° incident angle was used. The analysis was performed in an area of 200 × 200 μm² corresponding to 256 × 256 pixels. Charge compensation with an electron flood gun was implemented during the analysis and the positive ion spectra were calibrated by the C⁺, CH⁺, C₂⁺, and C₂H₃⁺ peaks.