Smartbeam 2 2 khz laser

Additional MALDI imaging experiments were performed to aid in lipid and metabolite annotation [34 (link),47 (link)] using a SolariX XR 7T-ESI/MALDI-Fourier-transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam II 2 kHz laser. All imaging experiments were performed at a lateral resolution of 100 µm, using a total of 100 laser shots per pixel with a small laser spot size. Data were collected in positive ionization mode across the m/z range of 300–1000 using 2 million data points, while negative MS spectra were collected over the m/z range of 100–850 using 1 million data points. Ion transmission voltage parameters were set as follows—funnel RF amplitude 120.0 Vpp, RF amplitude TOF 350.0 Vpp, TOF 0.6 ms, and RF frequency transfer optic 4 MHz. All the methods were externally calibrated through the electrospray ion source and NaTFA clusters. Internal calibration was applied using different lock masses—the [PC (16:0/18:1) + H]⁺ (m/z 760.58508), the [PC (16:0/18:1) + Na]⁺ (m/z 782.56702), and [PC (16:0/18:1) + K]⁺ (m/z 798.54096) were used for the MALDI-positive FT-ICR MSI analysis, and the 9-AA cluster ion (m/z 193.0771) for the MALDI-negative FT-ICR MSI experiments.

MALDI-MSI analysis was performed using a solariX 7 T 2ω MALDI-FTICR MS system (Bruker Daltonics), equipped with a Smartbeam II 2 kHz laser, operated in positive ionization mode and calibrated with red phosphorus before analysis. An abundant phosphatidylcholine ion (PC (34:1)+K⁺, mass-to-charge (m/z) 798.540964 was used for online calibration during data acquisition. For MSI of neuropeptides, the lateral resolution was 100 µm and 100 laser shots were collected at each position. Data were acquired at m/z range of 450–5000 using a Q1 value of 650 m/z. The transfer optics time-of-flight and frequency were 1 ms and 4 MHz, respectively. For MSI of dopamine and L-DOPA, the lateral resolution was 150 µm and spectra were collected by firing 100 laser shots per pixel. The scanned m/z range was 150-1500 and the Q1 value was 378 m/z. The transfer optics time-of-flight and frequency were 0.7 ms and 4 MHz, respectively. Data were visualized using FlexImaging software (Bruker Daltonics, version 4.1). The spectra were subjected to root mean square (RMS) normalization.