A major advantage of MALDI MS profiling and imaging over other MS-based methods is that molecules of interest remain in their natural environment, preserving their spatial relationship across the tissue section. In order to better understand disease mechanisms and resolve ambiguous diagnoses, we are developing methods to improve the anatomic pathology workflow and decrease the lateral resolution for routine histology-directed IMS to 10-50 µm. To date, we have identified several IMS-compatible histological stains for visualizing and targeting cells by IMS, such as methylene blue, cresyl violet, Giemsa, Alcian blue, and periodic acid Schiff. Continuing efforts to achieve 10-50 µm spatial resolution for routine histology-directed IMS are focused on spray coating technologies.
Spatially Targeted Liquid Micro-extractions for Protein Identification
Liquid extraction surface analyses (LESA) provide an efficient and spatially-targeted approach for identifying proteins directly from tissue surfaces. This workflow merges the power of histology-directed imaging experiments and the identification capacity of traditional proteomics.
Figure 1. An example of top-down protein identification from LESA extractions. Modified from: Rapid Commun Mass Spectrom 32 (5): 442-450.
Pathology interface for the molecular analysis of tissue by mass spectrometry
We are developing the Pathology Interface for Mass Spectrometry (PIMS) information management system. Major benefits provided by PIMS include:
- Distant collaborators can exchange information in the form of images, attachments, comments, disease classifications, and annotations using a custom browser-based web interface.
- Pathologists can view and annotate samples at 40X magnification.
- PIMS automatically registers annotated histology images and unstained serial sections.
- PIMS tracks research projects, samples, and protocols.
PIMS is the central touchstone for collaborators to direct analytical efforts within the Resource. Currently there are dozens of projects involving numerous investigators providing feedback.
Imaging mass spectrometry (IMS) generates molecular images directly from tissue sections to provide better diagnostic insights and expand the capabilities of clinical anatomic pathology. Although IMS technology has matured over recent years, the link between microscopy imaging currently used by pathologists and MS-based molecular imaging has not been established. We adapted the Vanderbilt University Tissue Core workflow for IMS into a web-based system that facilitates remote collaboration. The platform was designed to perform within acceptable web response times for viewing, annotating, and processing high resolution microscopy images. We describe a microscopy-driven approach to tissue analysis by IMS. The Pathology Interface for Mass Spectrometry is designed to provide clinical access to IMS technology and deliver enhanced diagnostic value.
Figure 2. Screenshots from the pathology interface for mass spectrometry interface highlight the software features and workflow. (a) A scanned H and E stained tissue section can be viewed through the image markup screen and examined remotely by a pathologist. (b) The pathologist can then annotate the tissue via a user-defined color palette to distinguish regions of interest, for example, tissue classes (e.g., normal versus disease) or cell types. Annotations can be made as discrete spots (as shown) or regions. (c) Pathology interface for mass spectrometry can zoom to regions of interest. Scale bars and the magnification level are automatically rescaled according to the zoom level. (d) A mass spectrometry spectrum acquired from the spot labeled “1” in panels b and c. Abstract and Figure: Norris et al., Pathology interface for the molecular analysis of tissue by mass spectrometry. J Pathol Inform, 2016.
Next Generation Histology-directed Imaging Mass Spectrometry Driven by Autofluorescence Microscopy
Histology-directed imaging mass spectrometry (IMS) is a spatially targeted IMS acquisition method informed by expert annotation that provides rapid molecular characterization of select tissue structures. The expert annotations are usually determined on digital whole slide images of histological stains where the staining preparation is incompatible with optimal IMS preparation, necessitating serial sections: one for annotation, one for IMS. Registration is then used to align staining annotations onto the IMS tissue section. Herein, we report a next-generation histology-directed platform implementing IMS-compatible autofluorescence (AF) microscopy taken prior to any staining or IMS. The platform enables two histology-directed workflows, one that improves the registration process between two separate tissue sections using automated, computational monomodal AF-to-AF microscopy image registration, and a registration-free approach that utilizes AF directly to identify ROIs and acquire IMS on the same section. The registration approach is fully automated and delivers state of the art accuracy in histology-directed workflows for transfer of annotations (∼3–10 μm based on 4 organs from 2 species) while the direct AF approach is registration-free, allowing targeting of the finest structures visible by AF microscopy. We demonstrate the platform in biologically relevant case studies of liver stage malaria and human kidney disease with spatially targeted acquisition of sparsely distributed (composing less than one tenth of 1% of the tissue section area) malaria infected mouse hepatocytes and glomeruli in the human kidney case study.
Registration software for this tool can be downloaded here.
Figure 3. Overview of the workflow for next generation histology-directed imaging. Abstract and graphical abstract: Patterson et al., Next Generation Histology-Directed Imaging Mass Spectrometry Driven by Autofluorescence Microscopy. Anal Chem, 2018.