Histological images used for mass spectrometry imaging analyzes

Gather classical histology and Mass Spectrometry Imaging: Here is the match Part-1

Introduction
In this application note, we will show the benefits of merging histomorphology 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 first part of this application note,
we’ll present four histological stainings which are listed in the table below with their target tissue and related therapeutic
area applications.
H&E
Haemotoxylin and Eosin, abbreviated as H&E, is the most common staining methods. It consists of two dyes with
different properties, a basic dye (Haemotoxylin) and an acidic dye (Eosin).
Eosin stains basic (or acidophilic) structures red or pink, whereas Haematoxylin is used to stain acidic (or basophilic)
structures purplish blue. For example, it is possible to distinguish the nucleus and parts of the cytoplasm containing the
RNA in purple from the rest of the cytoplasm stained in a different color (pink).

H&E discriminates histological strutures and supports MSI to validate molecular localization. H&E staining of a
rabbit eye section is used and shown in picture 1. The corresponding molecular image obtained by MSI is also presented
(MALDI-TOFMS, positive detection mode, 80 μm of spatial resolution). The image shows the localization of A2E
(N-retinyl-N-retinylidene ethanolamine, m/z 592.45) in the posterior part of the eye at the retina region level. A2E, a
major lipofuscin component, is a biomarker for cellular aging involved in some ophthalmic disease progression such as
age-related macular degeneration (AMD) taking place at the posterior part of the eye [2]. Different layers of the retina
region (sclera, choroid and the retina itself) are visualized in insert as well as 2AE distribution which is co-localized with
these small structures. A2E localization provides some evidence of its function in the retina.MSI can be used to study
A2E localization and modulation, thus its implication in retinal disease, such as macular degeneration.

Congo Red
Congo Red is a secondary diazo dye highlighting a strong no covalent binding affinity to cellulose fibers.
Conventional microscopy observation reveals a dull brick red coloration of amyloid plaque accumulation, elastic tissue,
eosinophil granules and a grey color for nuclei within tissue.
Congo Red is the most popular dye used as a probe for diagnosing amyloidosis. Amyloid plaques accumulation
within brain tissue is often linked to Alzheimer’s disease progression.

Nissl
Nissl or Cresyl Violet staining is commonly used to identify nervous structures within brain or spinal cord
tissue sections. Nissl staining marks the endoplasmic reticulum due to ribosomal RNA as well as the nucleus and other
accumulation of nucleic acid (Nissl bodies). It binds to negatively charged nucleic acids (RNA or DNA) which are
stained in purple-blue.
The picture 3 shows the Nissl staining of a sagittal brain section. Neuronal structures from Cerebellum or
Hippocampus region are well revealed and stained in purple blue. Granular Dental Gyrus and Cornu Ammonis layers
from hippocampus can be visualized as well as granular cells in the cerebellum (black arrows). High spatial resolution
MS images (MALDI-TOFMS, positive detection mode, 30 μm) obtained from these two regions are displayed. They
show two lipids distribution co-localized with histological nervous layers of the brain. Combining the use of Nissl and
the high spatial resolution MS Imaging of contrast, lipid ions can enhance the fine localization of drug or other markers
in the small brain structures to support drug activity or potential toxicity in a CNS study.

Methylene blue
Methylene blue is a heterocyclic aromatic chemical compound used to stain basophilic constituents of the tissue (DNA, RNA and carbohydrate polyanions) in blue.
Methylene blue enables the accurate visualization of beta cells in pancreatic islets as presented in picture 4
(black arrows). The less intensive blue color marks islets on the image. Molecular image of corresponding pancreas
section is also reported (MALDI-TOFMS, positive detection mode, 70 μm of spatial resolution). Insulin related ion
(m/z 5800.69) can be observed on mass spectrometric image and precisely correlated with Langerhans islet position on
methylene blue staining image [4]. In diabetes treatment, insulin analogues are used to rebuild the body’s natural
pattern of insulin release. The combination of methylene blue staining and MSI could validate the localization of these
insulin analogues and quantify their amount in a PK/PD study.

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

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