Fleximaging 5

Manufactured by Bruker

Sourced in Germany

FlexImaging 5.0 is a software application for mass spectrometry imaging. It provides tools for data acquisition, processing, and visualization of molecular images from tissue samples.

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FlexImaging (42 citations) FlexImaging version 5.0 (3 citations)

28 protocols using fleximaging 5

MALDI-MSI for Molecular Imaging

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MALDI-MSI experiments were carried out in positive reflectron mode over a mass range of m/z 200 to 1000 using a MALDI rapifleX tissuetyper (Bruker Daltonics) equipped with a 10 kHz Smartbeam 3D™ Nd:YAG laser. Data collected on the rapifleX was at a spatial resolution of 50 μm, summing up 500 laser shots/raster position.
FlexImaging 5.0 (Bruker Daltonics) software was used for initial data analysis. Normalization, molecular image extraction and spectral clustering were defined in SCiLS Lab 2018b (Bruker Daltronics) software typically using mass selection window of ±0.05 Da. MEDI9197 and heme were detected at m/z 594.4 and 616.1, respectively.

Mullins S.R., Vasilakos J.P., Deschler K., Grigsby I., Gillis P., John J., Elder M.J., Swales J., Timosenko E., Cooper Z., Dovedi S.J., Leishman A.J., Luheshi N., Elvecrog J., Tilahun A., Goodwin R., Herbst R., Tomai M.A, & Wilkinson R.W. (2019). Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies. Journal for Immunotherapy of Cancer, 7, 244.

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MALDI FTICR Imaging of N-Glycans

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As previously described, tissues were analyzed via imaging N-glycans using a MALDI FTICR mass spectrometer (SolariX Dual Source, 7T, Bruker Daltonics, m/z 500−5000). The data were then analyzed and visualized using FlexImaging 5.0 and SCiLS Lab 2017b (Bruker Daltonics). Finally, glycans were built and validated against the database in GlycoWorkbench, as well as built for graphical interpretation.^{28 (link),44 (link)}

West C.A., Liang H., Drake R.R, & Mehta A.S. (2020). New Enzymatic Approach to Distinguish Fucosylation Isomers of N‑Linked Glycans in Tissues Using MALDI Imaging Mass Spectrometry. Journal of proteome research, 19(8), 2989-2996.

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High-Resolution MALDI-MSI Imaging

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High-speed MALDI-MSI
acquisition was performed at 10 μm spatial resolution by using
a MALDI-TOF instrument (rapifleX, Bruker Daltonics). The MALDI source
is equipped with a scanning Smartbeam three-dimensional (3D) laser
featuring a laser beam diameter of 5 μm. Spectra were acquired
by using custom laser settings with a resulting field size of 10 μm.
The measurements were performed with the laser operating at a frequency
of 10 kHz with 20 laser pulses per pixel. Acquisition and subsequent
processing were performed by using the instrument software FlexImaging
5.0 (Bruker Daltonics). Acquisition of high-mass-resolution MSI data
was performed by using an Orbitrap Elite mass spectrometer (Thermo
Fisher Scientific GmbH, Bremen, Germany) coupled to a reduced-pressure
ESI/MALDI ion source (Spectroglyph LLC, Kennewick, WA). Further details
on the ion source can be found in the literature.^{12 (link)} The 349 nm MALDI laser (Spectra Physics, Mountain View,
CA) was operated at a repetition rate of 1000 Hz and pulse energy
of ∼1.5 μJ. The laser was focused to a spot size/step
size of ∼20 × 20 μm², mass resolution
was chosen to be 120,000 (at m/z 400), and the total scan time was 1.05 s/scan and pixel.

Michno W., Bowman A., Jha D., Minta K., Ge J., Koutarapu S., Zetterberg H., Blennow K., Lashley T., Heeren R.M, & Hanrieder J. (2024). Spatial Neurolipidomics at the Single Amyloid-β Plaque Level in Postmortem Human Alzheimer’s Disease Brain. ACS Chemical Neuroscience, 15(4), 877-888.

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Glycan Profiling by FTICR Mass Spectrometry

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A Solarix dual source 7T FT-ICR mass spectrometer (Bruker Daltonik, Bremen, Germany) containing a SmartBeam II laser operating at 2000 Hz was used for analysis of released N-glycan ions. Ions were detected with a laser spot size of 25 µm using 200 laser shots per pixel with a stepsize of 100 µm. N-glycans were measured in positive ion mode with a 1.2059 s transient over mass range 700-5000. Data was analyzed using FlexImaging 5.0 and SCiLS Lab 2019c (Bruker Daltonik) normalized to total ion current. Exported peak intensities were transformed using natural log prior to statistical testing. Glycans annotated by accurate mass were assigned by hexose content. Putative structures are described using databases from previous imaging studies on human tissues using GlycoWorkbench (58 (link), 60 (link)–67 (link)). Compositional accuracy of the glycan structures defined herein was determined based on accurate mass and prior structural characterizations by MALDI-FTICR MS, MALDI-TOF MS and LC-MS/MS (43 (link), 45 (link), 68 (link)).

Rujchanarong D., Scott D., Park Y., Brown S., Mehta A.S., Drake R., Sandusky G.E., Nakshatri H, & Angel P.M. (2022). Metabolic Links to Socioeconomic Stresses Uniquely Affecting Ancestry in Normal Breast Tissue at Risk for Breast Cancer. Frontiers in Oncology, 12, 876651.

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MALDI Imaging Mass Spectrometry Protocol

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MALDI MSI data were acquired with a rapifleX tissuetyper in single TOF mode (Bruker Daltonik GmBH, Bremen, Germany), equipped with a SmartBeam 3D laser. Mass spectra were the sum of 1000 individual laser shots, with a 90% laser intensity. Mass spectral peptidomic (m/z range 800 Da–5 kDa) images were obtained in positive reflector mode with a reflector voltage of 3005 V, a sample rate of 0.63 GS/s, a laser resolution of 50 µm and a raster width of 50 µm × 50 µm.
All the spectra are preprocessed with a Top Hat baseline algorithm for baseline subtraction and the resulting overall average spectrum of the ion image is TIC normalized in flexImaging 5.0 (Bruker Daltonik GmBH, Bremen, Germany) after recalibration in flexAnalysis 4.0 with external calibration standard. The results will be further processed in SCiLS lab 2016b (Bruker Daltonik GmBH, Bremen, Germany) and R software (Cardinal) [42 (link)].

Berghmans E., Van Raemdonck G., Schildermans K., Willems H., Boonen K., Maes E., Mertens I., Pauwels P, & Baggerman G. (2019). MALDI Mass Spectrometry Imaging Linked with Top-Down Proteomics as a Tool to Study the Non-Small-Cell Lung Cancer Tumor Microenvironment. Methods and Protocols, 2(2), 44.

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MALDI-MSI for Spatial Metabolite Profiling

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Optical images were acquired using a gel scanner at 1200 dpi resolution prior to fluorescence imaging and MALDI matrix application. Deposition of MALDI matrix (5 mg/mL DHB solution in MeCN:H₂O 60:40, 0.2% formic acid) was performed utilizing iMatrix sprayer in 20 passes with a spray density of 1 μL/cm². The matrix-coated slides were dried under vacuum for 60 min. MALDI-MSI data was acquired using a Bruker rapifleX MALDI Tissuetyper™ system operating in reflectron positive mode from m/z 200–2000 Da (Bruker Daltonik GmbH, Bremen, Germany). The smartbeam 3D laser was operated in a single scan mode setting (imaging −20 μm) and the image acquired using a pitch of 20 μm. The laser was operated at 10 kHz and 200 shots per pixel were recorded. Calibration was performed prior to MALDI-MSI data acquisition using red phosphorous deposited on the ITO slide. Instrument control was achieved via flexControl 4.0 (Bruker Daltonik GmbH, Bremen, Germany) and imaging acquisition was performed using flexImaging 5.0 (Bruker Daltonik GmbH, Bremen, Germany). Total ion current was utilized as normalization method. To display two-dimensional ion intensity maps, false colors are assigned to ions in the overall average mass spectrum in the flexImaging software or SCiLS Lab Pro 3D software (Bruker Daltonik GmbH, Bremen, Germany).

Cantrell T.P., Freeman C.J., Paul V.J., Agarwal V, & Garg N. (2019). Mass spectrometry-based integration and expansion of the chemical diversity harbored within a marine sponge. Journal of the American Society for Mass Spectrometry, 30(8), 1373-1384.

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MALDI-MSI Sample Preparation and Imaging

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For MALDI-MSI, ITO slides were sublimed with a thin layer of 1,5-DAN via a lab-built sublimation chamber. Prior to sublimation, slides were placed in a desiccator to come to room temperature for 15 min while the sublimation chamber was heated to 140 °C. Once at room temperature, the slides were weighed, and two slides were simultaneously sublimed within the chamber for 20 min, with pressure stabilizing at 105 mTorr for 1,5-DAN. Post sublimation, the slides were desiccated prior to a second weighing for determination of an average coverage, and 150 μg/cm² of 1,5-DAN was deposited. A digital scan was loaded into FlexImaging 5.0 (Bruker Daltonics, Bremen, Germany) to control the raster and acquisition of scans with spatial resolution set at 150 μm. The DESI method was utilized, and optimization of the instrument’s laser power was completed prior to imaging with 1000 shots per pixel at a 1 kHz repetition rate.

Zemaitis K.J., Izydorczak A.M., Thompson A.C, & Wood T.D. (2021). Streamlined Multimodal DESI and MALDI Mass Spectrometry Imaging on a Singular Dual-Source FT-ICR Mass Spectrometer. Metabolites, 11(4), 253.

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MALDI-TOF Tissue Imaging Workflow

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Frozen tissue sections were cut on a cryostat (CM1950, Leica Microsystems, Wetzlar, Germany) at a 10 μm thickness using indium-tin-oxide–coated glass slides (Bruker Daltonics, Bremen, Germany). Prior to washing, stored samples were placed in a vacuum chamber to dry. To remove endogenous lipids and inorganic salts, dried samples were immersed in 70% ethanol for 30 s, pure ethanol for 30 s, Carnoy’s solution for 3 min, pure ethanol for 30 s, 0.1% TFA for 1 min, and pure ethanol for 30 s. Prior to matrix coating, treated with a formic acid vapor. Sinapinic acid was used as a matrix. For mass spectrometric measurements, tissue areas were defined using the FlexControl 3.8 and FlexImaging 5.0 software packages (both Bruker Daltonics). Spectra were acquired using the rapifleX MALDI Tissuetyper (Bruker Daltonics) in positive linear mode, where ions were detected in a mass range of m/z 2000 to 20,000, with spatial resolution of 20 and 100 μm, respectively. A ready-made protein standard was used for spectra calibration (Bruker Daltonics). Visualization and statistical analysis were completed using FlexImaging and SciLS Lab 2016a (SCiLS, Bremen, Germany).

Kakuda N., Miyasaka T., Iwasaki N., Nirasawa T., Wada-Kakuda S., Takahashi-Fujigasaki J., Murayama S., Ihara Y, & Ikegawa M. (2017). Distinct deposition of amyloid-β species in brains with Alzheimer’s disease pathology visualized with MALDI imaging mass spectrometry. Acta Neuropathologica Communications, 5, 73.

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MALDI-FT-ICR MS Imaging of Microbial Colonies

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Mass spectrometry images were obtained as recently described (76 (link)) using an FT-ICR mass spectrometer (SolariX XR 9.4T, Bruker Daltonics, Bremen, Germany) mass calibrated from 200 m/z to 2,300 m/z to reach a mass accuracy of 0.5 ppm. A region of interest from agar microbial colonies was directly collected from the petri dish and transferred onto an indium tin oxide (ITO) glass slide (Bruker, Bremen, Germany) previously covered with double-sided conductive carbon tape. The samples were dried under vacuum and covered with an α-cyano-4-hydroxycinnamic acid matrix solution at 5 mg/mL (70:30 acetonitrile:water [vol/vol]). In total, 60 layers of α-cyano-4-hydroxycinnamic acid matrix were sprayed using a SunCollect instrument (SunChrom, Friedrichsdorf, Germany). FlexImaging 5.0 (Bruker Daltonics, Bremen, Germany) software was used for MALDI-FT-ICR MS imaging acquisition, with a pixel step size for the surface raster set to 100 μm.

Andrić S., Meyer T., Rigolet A., Prigent-Combaret C., Höfte M., Balleux G., Steels S., Hoff G., De Mot R., McCann A., De Pauw E., Argüelles Arias A, & Ongena M. (2021). Lipopeptide Interplay Mediates Molecular Interactions between Soil Bacilli and Pseudomonads. Microbiology Spectrum, 9(3), e02038-21.

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DESI-MALDI MSI Data Generation and Analysis

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All MSI images were generated in FlexImaging 5.0 (Bruker Daltonics, Bremen, Germany) with a 0.95 ICR noise threshold (default), with data reduced to 96.5%. Image generation of DESI-MSI was carried out by swapping the transient of serial acquisition from DESI into the file folder of the MALDI-MSI serial acquisition. This data file was post-processed with FTMS Processing 2.2.0 (Bruker Daltonics, Bremen, Germany), and the file was saved into a copy of the MALDI FlexImaging sequence subdirectory which was acquired, accounting for the proper number and alignment of pixels for each line scan of the completed DESI acquisition. This process is explained in greater depth in Supplementary Information. After creation of images, average spectra were created by importing into DataAnalysis 5.0 (Bruker Daltonics, Bremen, Germany) for both DESI and MALDI analyses. Identification of species was carried out in LIPID MAPS (www.lipidmaps.org/ (accessed on 17 February 2021)) [56 (link)], and METLIN (www.metlin.scripps.edu/ (accessed on 17 February 2021)) [57 (link)].

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