Fleximaging software

Automatic alignment of MALDI-MSI imaging data and H&E-section images was performed, using the Co-Register Image dialog in FlexImaging software (Bruker Daltonik, Bremen) (Ref.: PMID: 22011652). For three-dimensional visualization, representative MALDI-MSI images of selected masses were merged into the 3D-LSFM image z-stacks at the positions of the respective ROIs (Figs. 1e, 2e,l, Supplemental Video 1). Anatomical landmarks were used to align the positions and lateral dimensions of the (2D) MALDI-MSI planes with the 3D-LSFM reconstructions of the corresponding samples, using the Volume Fusion mode of the arivis Vision4D (vers. 3.0, arivis, Germany) software.

Species with the highest floral CNglyc content, and different distributions of CNglycs within tissues (based on dissection assays) were used for CNglyc visualisation by MALDI-MSI. Specifically, sections of G. robusta, M. tetraphylla, T. speciosissima and H. bucculenta florets were imaged. In addition, young fruits of N. kevedianus were opportunistically imaged as one of the species with high CNglycs in the ovary, and rachis cross sections of H. bucculenta, a species with high CNglycs in leaves and inflorescences, were imaged to examine the potential for CNglyc transport. Sections were imaged using the methods and instrument settings identical to those described in [17 (link), 18 ]. The area selected for imaging was defined using flexImaging software (v4.1, Bruker, Bremen, Germany). Data were imported into SCiLS lab software (version 2017a) where the respective Regions of Interest (ROIs) were selected, signal intensities of each region were exported and box-dot plots were generated.

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.

Fourty microns-thick transverse frozen sections were cut using a cryostat (Leica, Milton Keynes, UK) and fixed on a carbon-conductive adhesive tape which was in turn fixed on an indium tin oxide (ITO) slide (Bruker Daltonics, Bremen, Germany, cat no 237001).
All MSI measurements were performed using an Autoflex-Speed MALDI-TOF/TOF spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser (355 nm, 1000 Hz) and controlled using the Flex Control 3.4 software package. The mass spectrometer was operated with a negative polarity in the reflectron mode. Spectra were acquired in the mass range of m/z 100–600 for all (x, y) coordinates corresponding to the imaged tissue.
The laser raster size was set at 50 microns. The signal was initially optimized by manually adjusting the laser power and the number of laser shots fired. Accordingly, full-scan MS experiments were run by accumulating 400 satisfactory laser shots per raster position, and using the laser power leading to the best signal-to-noise ratio. Image acquisition was performed using the Flex Imaging 4.0 (Bruker Daltonics) software package. The correlation of the target plate with the optical image was performed from three distinct teaching points following the procedure of the Flex Imaging software (Bruker Daltonics).

MALDI-MS profiling spectra of the manually spotted tissue sections and imaging data of intact brain sections were acquired on an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a 200 Hz smart beam laser. The following parameters were adopted in the positive linear mode at a mass range of 3–25 kDa for spectral acquisition with a delayed extraction of 50 ns: ion source 1 voltage 20.00 kV, ion source 2 voltage 18.55 kV, lens voltage 6.80 kV and pulsed ion extraction 130 ns. Protein calibration standard I (Bruker Daltonics, Billerica, MA) was used to externally calibrate the instrument before data acquisition. Briefly, 2000 consecutive laser shots were accumulated for each deposited matrix droplet. The profiling spectra were smoothed and baseline subtracted using flexAnalysis (Bruker Daltonics, Billerica, MA). As for imaging, automated MSI acquisitions of the brain sections were controlled by flexImaging software (Bruker Daltonics, Billerica, MA). Array of spectra was collected at 200 µm intervals in both x and y dimensions, and each spectrum consists of 200 laser shots.