Histological images used for mass spectrometry imaging analyzes

Gather classical histology and Mass Spectrometry Imaging: Here is the match – Part 2

Introduction
In this application note, we will show the benefits of merging histological stains and Mass Spectrometry Imaging (MSI). The correlation between histological structures or disease related alterations and molecular species localization provides more information about biology or disease progression/mechanism taking place within tissue. In fact, histological features can drive molecular imaging interpretation and vice versa [1]. Some pathological changes are not visible by staining or immunohistochemistry (IHC) and MSI provides, in this case, a real added value in order to go beyond the visual inspection. In drug discovery, histological stains enable the visualization of biological tissue types, whereas MSI provides molecule localization and quantification and can also monitor disease state biomarkers level. Moreover, it is possible to evaluate toxicity induced by a treatment thanks to biomarkers quantification. These biomarkers can be spatially correlated with local tissue’s damages. In this second part of this application note, we’ll present four other histological stainings which are listed in the table below with their target tissue and related therapeutic area applications.

Periodic Acid-Schiff reaction (PAS)

The PAS stain is based on the action of Schiff reagent that reacts with molecular aldehyde groups. PAS stains carbohydrates and carbohydrate rich macromolecules in magenta for example glycogen, mucus in cells, basement membranes, brush borders of kidney tubules, or small and large intestine reticular fibers.

Azan Blue
Azan blue staining uses azocarmine B or G species followed by aniline blue to differentiate histological structures. Nuclei and erythro-cytes are stained in bright red whereas collagen basement membrane and mucin are stained in blue. Muscle and red blood cells highlight an orange to red color. The picture 2 shows a part of a mouse whole body section stained with Azan blue. Muscle layer can be clearly visualized from other structures such as adipose tissue or collagen. This staining can be useful to assess musculoskeletal disorders.

Moreover, MSI provides spatially preserved molecular information on specific metabolites or lipids contained in muscle fibers as presented in picture 3. This picture shows a transversal tissue section of a mouse back leg. Muscle fibers are highlighted in orange thanks to Azan blue staining. The distribution of two ribonucleotides, the adenosine monophos-phate (AMP, m/z 346.0584, 2a) and adenosine triphsosphate (ATP, m/z 505.9934, 2b) coincides with fiber-rich regions. Because these ribonucleotides are involved in muscle metabolism, combining Arzan blue staining with MSI enables to assess their distribution and modulation to study musculoskeletal or metabolic disorders.

Oil red 0
Oil Red O is a lysochrome diazo dye used to mark neutral triglycerides, lipids, fatty acids and lipoprotein. Lipids accumulation in tissue is stained in deep red. Nuclei stains blue/black.
Skin lipids are well observed on the Oil Red O staining image of the adipose tissue as presented in picture 4. The overlay distribution of two lipids reveals their typical localization in skin substructures. Adipose tissue differences are correlated with two lipids distribution, m/z 734,58 in red and m/z 758,58 in green respectively PC (32:0) and PC (34:1).Metabolic diseases and inflammation can be related to lipid synthesis dysfunction within discrete histological tissue. Combining Oil Red O and MSI of constitute lipids is a powerful approach to study lipid synthesis dysfunction in the skin, as well as in other tissues such as atherosclerotic tissues.

Masson’s trichrome
Masson’s trichrome is often used to stain connective tissue and to distinguish cellular items from extracellular items. Thus, nuclei and other basic-liking structures are stained in blue, cytoplasm, muscle, erythro-cytes and keratin are stained in bright-red. Then, nuclei is stained in black and collagen is stained green.
In picture 5 & 6, Masson’s trichrome is used to study a toxicity induced by a chemical compound in lung tissue. In fact, it reveals fibrotic area within lung tissue structure (airways and alveolus) thanks to green staining of collagen fibers as displayed. On molecular image, lysophosphatidic acid (LPA 16:0) ion ([M-H2O-H]-m/z 391.2253) is clearly distributed and concentrated in the fibrosis area. In this case, MSI aims to identify some molecular biomarkers of this phenomenon such as LPA. In the same time, MSI keeps regional speci-ficity and so explains this toxicity associated to atypical figures or biological processes. The content of collagen fibers will emerge thanks to the use of Masson’s trichrome combined to MSI.

Conclusion
Classical histology combined to MSI provides a valuable tool for the optimisation and validation of drug
targeting process.
Thus, we highlight that matchinghistological staining and MSI offers a real added value.

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