How to Prepare Samples for Spatial Metabolomics: The Essential Guide You Need
In spatial metabolomics, the success of mass spectrometry imaging (MSI) relies heavily on the quality of the samples prepared. However, preparing these samples involves a series of critical decisions that can greatly influence the outcome of your analysis. Researchers often face key questions: Should the samples be embedded, and if so, which embedding material is most suitable? What freezing method will best preserve the metabolic profile? These are just a few of the important considerations that can affect the reliability and accuracy of your results.
In this guide, we’ll explore the essential considerations for sample preparation, drawing insights from the study "Universal Sample Preparation: Unlocking Multimodal Molecular Tissue Imaging," published in Analytical Chemistry. This article introduces an innovative tissue sample embedding and processing method and assesses the method’s compatibility with several mass spectrometry imaging techniques, including Matrix-Assisted Laser Desorption/Ionization (MALDI), Desorption Electrospray Ionization (DESI), and Secondary Ion Mass Spectrometry (SIMS). By delving into this research, we’ll guide you through a comprehensive approach to sample preparation, from selecting the optimal embedding materials to fine-tuning freezing techniques. These insights will empower you to make informed decisions, ensuring high-quality, reproducible data for your spatial metabolomics studies.
To Embed or Not to Embed?
To assess the impact of embedding on tissue morphology, the study applied four different treatments: a) no embedding, b) embedding in OCT or the newly developed HPMC+PVP hydrogel blocks, c) freezing at -80°C, and d) snap-freezing and conducted H&E-staining experiments (Figure 1). The results revealed that embedding with HPMC+PVP followed by snap-freezing significantly improved tissue morphology compared to unembedded sections, with HPMC+PVP snap-frozen tissues showing superior tissue structure and staining properties.
Figure 1. Effects of embedding on rat renal cortex tissue morphology
The study further assessed the reproducibility of the HPMC+PVP snap-freezing method by using different MSI techniques on co-embedded kidney, liver, and spleen specimens (Figure 2). DESI-MSI imaging, performed at a high spatial resolution of 25 µm on adjacent regions, confirmed minimal displacement of polar analytes (such as GSH and FA (18:2)) while clearly delineating larger morphological features, such as liver zones and red and white pulp areas. Additionally, MALDI-MSI imaging at a spatial resolution of 10 µm provided detailed visualization of analyte distribution, revealing even smaller morphological features, such as the renal cortical tubular system.
Figure 2. Reproducibility evaluation of the optimized embedding protocol and achievable imaging resolution
Which Embedding Material Works Best?
To investigate the impact of different embedding materials on analyte behavior in MSI, the study evaluated various embedding media using DESI-MSI. The results (Figure 3) showed that HPMC + PVP-embedded snap-frozen tissue blocks exhibited minimal analyte displacement, whereas HPMC + PVP-embedded samples frozen at -80°C demonstrated significant migration of polar analytes into the embedding medium. Regardless of the freezing method, gelatin showed considerable displacement of both polar molecules and structural lipids. Na-CMC demonstrated analyte displacement similar to HPMC+PVP; however, deposition of the embedding medium on the sample surface resulted in partial ion suppression (shown as darker shadows in the image), and the background staining from the embedding medium was notably strong in H&E-stained sections. HPMA facilitated the displacement of polar analytes, consistent with observations in positive ion mode. HPMC snap-frozen samples showed minimal displacement of adenosine monophosphate (AMP) and glutathione (GSH), while samples frozen at -80°C exhibited significant leakage.
Figure 3. Evaluation of different embedding media by DESI-MSI
The authors also compared various parameters of the embedding media used in this study, including preparation complexity, viscosity, tissue adherence, and matrix interference. The results indicated that while the preparation of HPMC+PVP is somewhat more complex, it excels in other key properties, making it a relatively ideal embedding medium.
Table 1. Summary of sectioning properties for the evaluated embedding media.
Snap Freezing or Freezer Freezing?
To evaluate the impact of different freezing methods on MSI, the study investigated the performance of samples with various embedding media subjected to different freezing techniques using DESI-MSI. The results (Figure 3) demonstrated that, compared to slow freezing in a -80°C freezer, snap-freezing resulted in minimal displacement of polar analytes across all embedding media, with the exception of phospholipids, which exhibited minimal displacement regardless of the freezing method. For example, HPMC + PVP-embedded snap-frozen tissue blocks showed almost no analyte displacement, whereas samples frozen in a -80°C freezer exhibited significant migration of polar analytes into the embedding medium. Snap-frozen HPMC samples displayed minimal displacement of adenosine monophosphate (AMP) and glutathione (GSH), while samples frozen in the -80°C freezer showed considerable leakage.
In addition, H&E staining of tissue sections frozen using different methods revealed that snap-frozen tissues retained better tissue morphology and staining characteristics. In contrast, samples frozen in a -80°C freezer exhibited significant morphological damage, with H&E-stained sections showing prominent freezing defects, including tissue rupture and large cracks caused by ice crystal formation, which hindered microscopic analysis (Figure 1). This is attributed to the slower freezing process in the -80°C freezer, which takes approximately 15-20 minutes, compared to the rapid 1-2 minute snap-freezing method. The damage observed in the -80°C freezer samples was a result of the formation of large ice crystals during the prolonged freezing process, which disrupted tissue integrity. Based on these findings, snap-freezing is recommended for preparing tissue samples for MSI.
Step-by-Step Guide to Spatial Metabolomics Sample Preparation
MetwareBio offers an advanced spatial metabolomics service, delivering precise and comprehensive tissue analysis using cutting-edge technologies. Our service utilizes MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), a highly sensitive and versatile technique ideal for spatial metabolomics analysis. We provide a range of spatial resolutions, from 100 µm for larger tissue structures to ultra-high resolution at 5 µm, allowing for detailed exploration of metabolites across various biological contexts.
To ensure optimal results, MetwareBio also offers a detailed step-by-step guide for sample preparation. This reference guide helps ensure your tissue samples are properly processed, maximizing the accuracy and reliability of the spatial metabolomics analysis for your research.
I. Tissue Preparation:
Non-embedded tissues (e.g., brain, liver, kidney, heart, spleen, with low water content):
Collect tissue samples (not exceeding 26*76mm) while maintaining tissue integrity. Place the tissue in an aluminum foil boat with the cutting surface facing downward. Immerse the boat in liquid nitrogen for 15-30 seconds until the tissue turns white. Remove and store the sample at -80°C for transport on dry ice.
Embedded tissues (e.g., eyes, lungs, plant tissues, with higher water content):
Collect tissue samples (not exceeding 26*76mm), ensuring integrity. Prepare the embedding medium (1-2% carboxymethyl cellulose (CMC) or HPMC + PVP). Avoid OCT, as it can interfere with ionization during analysis. Add 1mL of embedding medium to the embedding box, ensuring no air bubbles, and position the tissue for sectioning. Cover the tissue completely with the embedding medium, then freeze the box in a dry ice and ethanol bath for 3-5 minutes. Once solidified, store at -80°C for transport on dry ice.
II. Tissue Sectioning
Sectioning Parameters: Section thickness should range from 8 to 20 microns, with a maximum size of 15*65mm. Mark sections clearly, leaving space between them, and provide 3-6 sections per sample.
Sectioning Procedure: After removing from the -80°C freezer, equilibrate tissue in a -20°C freezer or cryostat for 1 hour. Adjust the tissue orientation on the cryostat sample holder, then section according to the cryostat’s instructions. Transfer sections to pre-chilled slides with a cold brush. Melt the sections by pressing the back of the slide with your hand until transparent. Rub the back of the slide to remove moisture and turn the tissue white. Dry the slides in a vacuum for 20 minutes. Store at -80°C and transport on dry ice.
Note: Formalin-fixed, paraffin-embedded, or stained samples (e.g., H&E) may interfere with mass spectrometry and are not recommended for spatial metabolomics studies.
Reference
Dannhorn A, Kazanc E, Ling S, et al. Universal Sample Preparation Unlocking Multimodal Molecular Tissue Imaging. Anal Chem. 2020;92(16):11080-11088. doi:10.1021/acs.analchem.0c00826
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