Imagequest software

Manufactured by Thermo Fisher Scientific

Sourced in United States

ImageQuest software is a digital image acquisition and analysis tool developed by Thermo Fisher Scientific. It provides an intuitive interface for capturing, managing, and analyzing images from various imaging devices used in scientific research and laboratory settings.

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Lab products found in correlation

13 protocols using imagequest software

MALDI-MSI Data Processing Protocol

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Each MALDI‐MSI experiment comprised multidimensional data whereby every pixel (x, y coordinate) was associated with a mass spectrum (m/z, intensities). Data from MALDI‐MSI were converted to imzML format for processing.28 Using an in‐house R script, ions were retained above a threshold intensity and the m/z ratio of ions detected were summed across integral regions of 10 ppm to allow for alterations in m/z mass accuracy across the experiment (“binning”). Ion intensities were normalized to total ion count, mean centered, and Pareto scaled before production of single ion images or multivariate statistical analysis (PCA). During PCA, pixels were considered analogous to observations, and m/z values were considered analogous to variables. Principal components loadings were normalized according to their contribution to the overall variation before computing averages from different experiments. Multi‐ion image overlays were produced using ImageQuest software (Thermo Fisher Scientific).

Hall Z., Bond N.J., Ashmore T., Sanders F., Ament Z., Wang X., Murray A.J., Bellafante E., Virtue S., Vidal‐Puig A., Allison M., Davies S.E., Koulman A., Vacca M, & Griffin J.L. (2017). Lipid zonation and phospholipid remodeling in nonalcoholic fatty liver disease. Hepatology (Baltimore, Md.), 65(4), 1165-1180.

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MALDI-MSI Imaging of Tissue Samples

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A Thermo Scientific LTQ XL linear ion trap mass spectrometer (MALDI LTQ XL) was used for MALDI-MSI experiments.²³ The MALDI source on this instrument is an intermediate-pressure source (7.5 × 10⁻² Torr) with a pulsed nitrogen laser at 337.7 nm, a frequency of 60 Hz and energy of 250 μJ per pulse at 100% laser power. Sectioned tissue was dried in the desiccator for ~1 hour under vacuum and spray coated with dihydroxybenzoic acid (DHB) matrix at 40 mg/ml in 70:30 MeOH:H₂O with 10mM sodium acetate and 0.1% trifluoroacetic acid (TFA) using a Meinhard nebulizer. MALDI data were acquired using a laser energy and step size of 2.5 μJ and 100 μm, respectively. To assess background interference a section of the gelatin-based mold was mounted onto a glass slide and coated with matrix under the same conditions as the tissue. In addition a control slide was also coated with DHB matrix for comparison. All imaging data was analyzed using ImageQuest Software (Thermo Scientific).

Gill E.L., Yost R.A., Vedam-Mai V, & Garrett T.J. (2016). Precast Gelatin-Based Molds for Tissue Embedding Compatible with Mass Spectrometry Imaging. Analytical chemistry, 89(1), 576-580.

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Mass Spectrometry-Based Compound Identification

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The obtained MS/MS spectral data from standards and samples were submitted to structural analysis with Mass Frontier software (v. 6.0, Thermo Scientific, California, USA). The inputted structures are analyzed using algorithms and database information to produce fragment possibilities, which are then compared to the MS/MS spectra to assist in compound identification. Chemical images were treated with ImageQuest software (Thermo Scientific, California, USA) and all intensities were normalized according to the total ion current.

de Oliveira D.N., Ferreira M.S, & Catharino R.R. (2014). Rapid and Simultaneous In Situ Assessment of Aflatoxins and Stilbenes Using Silica Plate Imprinting Mass Spectrometry Imaging. PLoS ONE, 9(3), e90901.

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MALDI-MSI Analysis of Gd-based Contrast Agents

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MALDI-Mass spectrometry imaging (MALDI-MSI) was performed in positive
ion mode on a MALDI LTQ Orbitrap XL mass spectrometer (Thermo Scientific,
Waltham, MA, USA) equipped with a N2 laser. LTQ Tune software (Thermo
Scientific) was used to select the imaging region and step size, and Xcaliber
(Thermo Scientific, Waltham) was used to select the instrument parameters.
Imaging was performed on three control mice and three mice dosed with the
Gd-NM600 compound at 75 μm raster step size, from 130–2000 mass to
charge ratio (m/z) at 60K resolution. Two microscans were averaged at each
pixel. ImageQuest software (Thermo Scientific) was used to view raw data and
export the raw data to an imzML format. MSiReader software (1 (link)) was used to generate images unique to mice dosed
with the compound (experimental mice). Briefly, m/z elements that were present
in at least 10% of the interrogated zone (experimental mice) and in less than 5%
of the reference zone (control mice) or in over 5% of the reference zone with a
ratio greater than 2 were selected. Images for these m/z were then identified
and manually inspected.
For MALDI mass spectra of Gd-DOTA, and Gd-BOPTA (MultiHance), a mixture
of these compounds formulated at 1 μmol in distilled water and
2,5-dihydroxybenzoic acid (DHB, Acros Organics) matrix (40 mg/mL) was used to
obtain the MALDI mass spectra.

Zhang R.R., Choi C., Brunnquell C.L., Hernandez R., Pinchuk A.N., Grudzinski J.G., Clark P.A., McMillan A.B., Audhya A., Jeffrey J., Kuo J.S, & Weichert J.P. (2022). Next Generation Cancer Magnetic Resonance Imaging with Tumor-Targeted Alkylphosphocholine Metal Analogs. Investigative radiology, 57(10), 655-663.

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MALDI-IMS for Gemcitabine Mapping in Tumors

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MALDI-IMS experiments were performed as previously described (14 (link)). In brief, BxPC3 tumor xenografts were
removed and flash-frozen in liquid nitrogen before storage at
−80°C. Frozen tissue samples were then cut into 12 μm thick
sections using a cryostat (Leica Microsystems, Buffalo Grove, IL). These frozen
sections were then thaw-mounted onto gold-coasted, stainless steel MALDI target
plates. Matrix solution (2’,4’,6’-trihydroxyacetophenone,
20 mg/mL in 60:40 methanol:water with 0.1% trifluoroacetic acid) was manually
applied to tissues using a glass nebulizer. MALDI mass spectra were acquired
using a linear ion trap (LTQ XL) mass spectrometer (Thermo Fisher Scientific) in
MS/MS mode. MS/MS was performed on the protonated parent ion of gemcitabine (m/z
264) and full product ion spectra were obtained, allowing GEM to be
differentiated by pseudo-selected reaction monitoring. The main fragment ion of
gemcitabine at m/z 112 was utilized for image reconstruction. Spectra were
obtained for each section at 150 μm spatial resolution. Images were
generated using ImageQuest software (Thermo Fisher Scientific) by analyzing the
main fragment ion intensity as a function of the position over the tissue
surface. For statistical comparisons, average spectra were generated over each
tissue section and the intensities of m/z 112 were exported for further
comparisons.

Dosch A.R., Dai X., Reyzer M.L., Mehra S., Srinivasan S., Willobee B.A., Kwon D., Kashikar N., Caprioli R., Merchant N.B, & Nagathihalli N.S. (2020). Combined Src/EGFR inhibition targets STAT3 signaling and induces stromal remodeling to improve survival in pancreatic cancer. Molecular cancer research : MCR, 18(4), 623-631.

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Mass Spectrometry Imaging of Tenofovir and Metabolites

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Flash-frozen dermal tissue samples collected adjacent to the TAF implants and sectioned were analyzed with a linear ion trap mass spectrometer equipped with a MALDI source and a nitrogen laser (LTQ XL, Thermo Scientific, Waltham, MA). The structures of compounds of interest are shown in Scheme 1. Targeted MS/MS methods were optimized for each compound using authentic samples as standards, with the final parameters shown in Appendix A under Supplementary Information. Pseudo-selected reaction monitoring mode was used for imaging by isolating precursor ions with a 1 Da window, fragmenting them, and acquiring full product ion mass spectra at each pixel. TFV, TFV-MP, and TFV-DP were analyzed in negative ion mode, while TAF, Metabolite X, and Metabolite Y were analyzed in positive ion mode. In most cases, after imaging, the matrix was removed and the tissue section that had been imaged was H&E stained for better image registration with tissue morphology. Standard images were acquired at either 50 or 100 µm spatial resolution. Reconstructed ion images were generated in ImageQuest software (Thermo Scientific, Waltham, MA) by plotting the intensity of the diagnostic fragment ion or ions as a function of location across the tissue surface.

Gunawardana M., Remedios-Chan M., Sanchez D., Webster S., Castonguay A.E., Webster P., Buser C., Moss J.A., Trinh M., Beliveau M., Hendrix C.W., Marzinke M.A., Tuck M., Caprioli R.M., Reyzer M.L., Kuo J., Gallay P.A, & Baum M.M. (2022). Fundamental aspects of long-acting tenofovir alafenamide delivery from subdermal implants for HIV prophylaxis. Scientific Reports, 12, 8224.

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MALDI-MS Imaging of Tissue Sections

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Mass spectrometry imaging experiments were carried out on serial sections taken before and after those collected for LCM. Sections were taken at 10 μm using a CM1810 cryostat (Leica), thaw-mounted onto Superfrost Plus glass slides (Thermo Fisher Scientific), and stored at −80°C until analysis. Upon analysis, slides were removed from the −80°C freezer and desiccated for 15 min prior to matrix deposition. 2,5-dihydroxybenzoic acid (DHB) (25 mg/ml in 50% methanol containing 0.15% trifluoroacetic acid [TFA]) was deposited over the tissue sections using the HTX sprayer (HTX). Spray parameters were as follows: 60°C, flow rate of 50 μl/min, velocity of 1,200, 8 lb/in², and 30 cycles. Imaging acquisition was carried out using a MALDI LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific) operated in the positive ion mode with a mass range of m/z 185 to 400 and a pixel resolution of 75 μm. All data were processed using ImageQuest software (Thermo Fisher Scientific). Following data acquisition, the matrix was washed from the slides using 70% ethanol, and the sections were stained with hematoxylin and eosin (H&E) according the manufacturer’s protocol (Thermo Fisher Scientific).

Egbelowo O., Sarathy J.P., Gausi K., Zimmerman M.D., Wang H., Wijnant G.J., Kaya F., Gengenbacher M., Van N., Degefu Y., Nacy C., Aldridge B.B., Carter C.L., Denti P, & Dartois V. (2021). Pharmacokinetics and Target Attainment of SQ109 in Plasma and Human-Like Tuberculosis Lesions in Rabbits. Antimicrobial Agents and Chemotherapy, 65(9), e00024-21.

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MALDI Imaging of Bioactive Compounds

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A 50 mg/mL solution of 2,5-dihydroxybenzoic acid in a methanol: water mixture (7:3, v/v) served as a matrix. The matrix solution was sprayed uniformly over the slices by means of an airbrush with a 0.2 mm caliber nozzle (Procon Boy FWA Platinum; Mr. Hobby, Tokyo, Japan). MSI was performed using a MALDI TOF/TOF-type instrument (Autoflex) and the LTQ-XL linear ion trap mass spectrometer. Autoflex was used for imaging based on the MS data, and LTQ-XL was mainly used for imaging based on tandem mass spectrometry (MS/MS) data. This was done because Autoflex is suitable for simultaneous analysis of multiple samples, whereas LTQ-XL can perform simultaneous imaging on the basis of MS and MS/MS data. The data were acquired with a step size of 50 µm in both analyses. The FlexImaging software 4.0 (Bruker Daltonics) and ImageQuest software (Thermo Fisher Scientific) were used to create two-dimensional ion density maps. Normalization of the spectra to the total ion current was also performed in the imaging software. The distribution of the MS/MS product ions was visualized without normalization.

Zaima N., Kinoshita S., Hieda N., Kugo H., Narisawa K., Yamamoto A., Yanagimoto K, & Moriyama T. (2016). Effect of dietary fish oil on mouse testosterone level and the distribution of eicosapentaenoic acid-containing phosphatidylcholine in testicular interstitium. Biochemistry and Biophysics Reports, 7, 259-265.

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Quantitative Mass Spectrometry Imaging Analysis

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Spectral and MS/MS results were analyzed using Mass Frontier software (v. 6.0, Thermo Scientific, San Jose, CA). MSI data were treated using ImageQuest software (Thermo Scientific, San Jose, CA); quantification using MSI was carried out according to de Oliveira et al.20 , where the area (pixels) of the obtained chemical images in grayscale were analyzed using the ImageJ software (National Institutes of Health – Open Source). This software provided arbitrary values regarding pixel intensity: a lighter color in the scale meant less amount of the substance, while a darker color meant a higher amount, much like an HPLC approach, where larger peaks signify larger amounts of the analyte. No data normalization was performed. The obtained arbitrary values provided by ImageJ were then used to plot the analytical curve.

de Menezes M., de Oliveira D.N, & Catharino R.R. (2016). Capillary-induced Homogenization of Matrix in Paper: A Powerful Approach for the Quantification of Active Pharmaceutical Ingredients Using Mass Spectrometry Imaging. Scientific Reports, 6, 29970.

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MALDI Imaging Data Analysis Protocol

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The MALDI images were generated using the ImageQuest software (Thermo Scientific, San Jose, CA, USA). With this software, a m/z range is plotted for signal intensity for each pixel (mass spectrum) across a given area (tissue section). The quality of the images was improved during the image creation process by selecting the m/z range of interest and doing a normalization as a ratio of total ion current (TIC) for each mass spectrum. Different regions of interest (ROI) were analyzed including hippocampus, cortex, amygdala, cerebellum, and striatum. The spectra intensity was further normalized as a ratio of the peak or m/z value with the highest intensity, PC[(34 + 1) + K]⁺ in positive ion mode and PI[18:0/20:4]⁻ in negative ion mode and the average was calculated using the OriginPro 8 software. The most intense peak was considered the 100% and the intensity of the rest of the peaks was calculated as a percentage. The two-tailed unpaired Student’s t-test was used for the comparison of two groups. The results were considered significant when p ≤ 0.05.

González de San Román E., Llorente-Ovejero A., Martínez-Gardeazabal J., Moreno-Rodríguez M., Giménez-Llort L., Manuel I, & Rodríguez-Puertas R. (2021). Modulation of Neurolipid Signaling and Specific Lipid Species in the Triple Transgenic Mouse Model of Alzheimer’s Disease. International Journal of Molecular Sciences, 22(22), 12256.

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