Lipidomic Analysis of Mouse Brain to Evaluate the Efficacy and Preservation of Different Tissue Preparatory Techniques by IR-MALDESI-MSI

Abstract

Numerous preparatory methods have been developed to preserve the cellular and structural integrity of various biological tissues for different -omics studies. Herein, two preparatory methods for mass spectrometry imaging (MSI) were evaluated, fresh-frozen and sucrose-embedded, paraformaldehyde (PFA) fixed, in terms of ion abundance, putative lipid identifications, and preservation of analyte spatial distributions. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI)-MSI was utilized to compare the preparatory methods of interest with and without the use of the conventional ice matrix. There were 2.5-fold and 1.6-fold more lipid species putatively identified in positive-and negative-ion modes, respectively, for sucrose-embedded, PFA-fixed tissues without an ice matrix relative to the current IR-MALDESI-MSI gold-standard, fresh-frozen tissue preparation with an exogenous ice matrix. Furthermore, sucrose-embedded tissues demonstrated improved spatial distribution of ions resulting from the cryo-protective property of sucrose and paraformaldehyde fixation. Evidence from these investigations supports sucrose-embedding without ice matrix as an alternative preparatory technique for IR-MALDESI-MSI.

Oversampling for Enhanced Spatial Resolution of Zebrafish by Top-Hat IR-MALDESI-MSI

“…The head of the zebrafish was targeted for these experiments while using an m / z range of 300–600. No ice matrix was applied for these tissues, as sucrose-embedding was performed and acted as an energy absorbing matrix. , After spot size characterization, adjacent sections were run in the following order: (1) 140 μm × 125 μm (RP = 240 000 fwhm at m / z 200), (2) 20 μm × 20 μm, (3) 40 μm × 40 μm, (4) 60 μm × 60 μm, and (5) 140 μm × 125 μm (RP = 120 000 fwhm at m / z 200) (Figure C). The oversampling experiments on zebrafish (i.e., 20 μm × 20 μm, 40 μm × 40 μm, and 60 μm × 60 μm) produced square spot shapes of the same width and height; therefore, they will be referred to as 20 μm, 40 μm, and 60 μm from this point forward.…”

“…For UV-MALDI, the laser is complemented with an organic matrix co-crystallized with the sample, where energy is absorbed at the same or similar wavelength as the laser (e.g., 337 or 355 nm) . In the case of IR-MALDESI, ice and sucrose-embedding , have been utilized as the energy absorbing matrix, as the laser wavelength for this platform is 2970 nm. Because a smaller wavelength is commonly utilized for MALDI systems, they produce smaller spot sizes compared to IR-MALDESI .…”

Maximized Spatial Information and Minimized Acquisition Time of Top-Hat IR-MALDESI-MSI of Zebrafish Using Nested Regions of Interest (nROIs)

“…Nonsequential mouse liver sections were cut to 10 μm thickness immediately prior to analysis using a Leica CM1950 cryostat (Buffalo Grove, IL) at −16 °C. The Kullman lab euthanized the healthy adult female zebrafish by cold water and conducted the sucrose-embedding procedure to preserve tissue morphology . This involved a small ventral incision followed by 4% paraformaldehyde fixation for 1 week at 4 °C.…”

“…The Kullman lab euthanized the healthy adult female zebrafish by cold water and conducted the sucrose-embedding procedure to preserve tissue morphology. 27 This involved a small ventral incision followed by 4% paraformaldehyde fixation for 1 week at 4 °C. Then, the fish was washed three times in phosphate-buffered saline and placed in a 30% sucrose solution overnight at 4 °C prior to CMC embedding.…”