Mass Spectrometry Imaging (MSI) is a molecular imaging technique in which target molecules are ionized on a tissue section, and the distribution of ions are determined with a mass spectrometer and visualized. This technique is also called imaging mass spectrometry (IMS). In MSI, a laser beam is focused at regular intervals (tens to hundreds of micrometers) on frozen tissue sections, and the ions that arise at those points are directly detected with an ultra-high resolution mass spectrometer. Therefore, this technique does not require any labeling to target compounds. As this technique can monitor more than two compounds simultaneously, it is a very powerful technique which can analyze metabolites and biomarkers in addition to target compounds simultaneously. As the space between the laser spots can be adjusted in the range of tens to hundreds of micrometers, it is suitable for evaluating localization in micro areas in a heterogeneous structure such as ocular tissues and tumor tissues.
MSIH&EMSI / H&E
Case study of application area and analysis by MSI
MSI in drug development
It is very important in drug development to visually monitor in-vivo pharmacokinetics and distribution of dosing drugs to specific tissues and local areas to understand the characteristics of drugs. Autoradiography with radioisotopes (RI) has been conventionally used as a precise method for evaluating distribution in the body; however, it is difficult for this method to detect unaltered compounds and their metabolites separately. MSI is expected to be used as a complemental method to autoradiography in drug development research since it can distinguish unaltered compounds and metabolites separately.
By merging with images of hematoxylin-eosin staining and immunohistochemical staining from optical microscopes, localization of compounds can be observed in addition with morphological and pathological characteristics and confirm the extent to which the compounds have reached their target tissues. MSI is expected to be utilized in evaluation of drug efficacy in target tissues, confirmation of Proof of Concept (POC), and clarification of mechanism of toxicity expressions.
Low molecular compounds, nucleic acid medicines, and peptide pharmaceuticals are possible examples of target drugs that can be analyzed. Applications are not limited to just the pharmaceutical field but have a wider range such as various samples and compounds in the food and agrochemical fields.
Nat. Med., 2001, 7: 493-496
Flow of MSI
We employ Matrix Assisted Laser Desorption Ionization (MALDI) as an ionization method.
Experiment flow of MALDI-MSI
Animal experiment
Resection and freezing of target tissue
Preparation of frozen section
Spraying of matrix as an ionization assisting agent onto the frozen section slice adhered onto the glass slide dedicated to MSI
Analysis with MSI equipment
Laser beam is focused onto a given area at regular intervals on the section slide. Two modes of analysis, “whole analysis” with wide intervals to understand the distribution of the whole tissue and “high resolution analysis” with narrow intervals to see the distribution in detail.
We have introduced solariX from Bruker as our MSI equipment. It is capable of accurate mass spectrometry at the world’s highest level of ultra-high resolution which makes it possible to determine the molecular weights of target compounds accurately.
solariX
Case study: Evaluation of chloroquine localization in the eyeball of pigmented rats
Chloroquine
MSI was used in the evaluation of localization of chloroquine in rat eyeballs.
In past research, it has become known through autoradiography in rats that administered chloroquine accumulates in the uvea (retina). Vacuolation of retinal nerve fiber layer and hyperplasia of pigmented epithelium have been observed as toxicity effects. However, detailed distribution of chloroquine in those regions had not been clarified.
Chloroquine was administered to pigmented rats by single oral dosing (6, 20, 100 mg/kg), and the eyeballs were resected on the day after dosing. Frozen sections were prepared from the resected eyeballs, and whole analysis and high-resolution analysis of the ocular tissues were conducted by MSI.
Quantitative analysis can be conducted by analyzing chloroquine standard solutions of known concentrations.
Chloroquine signals were detected near the retina in all treatment groups
Quantitation regions were selected in the retinal outer layer (A) and retinal inner layer (B) in each eyeball (two regions for each layer). Mean of the results are shown below.
MSI quantitative value (μg/g,mean)
No.
A：Rerinal outer layer
B：Rerinal inner layer
101
<LLOQ
<LLOQ
103
12.6
<LLOQ
105
79.8
4.02
107
271
43.9
LLOQ : 1 µg/g
In addition to analyzing the whole tissue section, high-resolution analysis of selected regions is also possible. By high-resolution analysis with laser irradiation every 15 micrometers in the yellow squared area, it was found that chloroquine was mainly localized in the pigmented epithelium and choroid outside of it.
By comparing the distribution image of chloroquine, distribution image of A2E (specific marker of pigmented epithelium on retinal cell layer), and HE stain image of the section after MSI analysis, it was confirmed chloroquine was distributed in the choroid and pigmented epithelium.
