The 8th Mass Spectrometry CVG Annual Symposium - Full Day Event!!!
Presentation No. 9 - From Single Cells to Whole Body Sections: Multiscale Imaging of Phospholipids by MALDI MS
Pierre Chaurand1; Peggi Angel2; Richard M. Caprioli2
1Presently at University of Montreal, Canada 2Vanderbilt University, Nashville , TN
Novel Aspect: High resolution imaging MS of phospholipids from tissue sections down to cellular dimensions
Introduction: For the past decade, MALDI MS has been used to image the composition and spatial arrangement of biomolecules in thin tissue sections. One of the major challenges of the technology is to image features down to the dimension of a single cell. This is only possible after both instrumentation (laser focus) and sample preparation (matrix disposition) considerations. We have recently built and characterized a MALDI source capable of focusing laser light on regions <5 µm in diameter. In parallel, advances in matrix deposition via sublimation allow generation of homogenous matrix films on sections. Using this sample preparation strategy, we report MALDI MS imaging of phospholipid (PL) distribution on a variety of tissue sections with spatial resolutions ranging from 5-200 µm.
Methods: Sections were cut at 10-12 µm from a freshly frozen 2-day old mouse pup, adult mouse brain, or adult mouse heart and thaw-mounted on conductive target plates. Serial sections were also cut and H&E stained. Matrix (2,5-DHB) deposition was performed using a sublimation apparatus coupled to a rough vacuum pump (5x10-2 Torr) and heated to ~100ºC. MS data were acquired by MALDI-TOF MS using either a Bruker Ultraflex II mass spectrometer operated in reflectron mode. High spatial resolution (5-25 µm) data was acquired on a custom-built MALDI-TOF instrument operated in linear mode. Ion images were assembled using FlexImaging or Biomap software.
Results: Tissue sections destined to be analyzed by IMS come in a wide range of dimensions with different analytical requirements. Whole body sections (from mouse, rat, or other species) are typically surveyed at low spatial resolution to obtain global distributions of molecular content. Small tissue sections or specific anatomical features may require acquisition of higher resolution data down to the cellular level to obtain biological information distinct to a feature or pathology.
For all tissue sections, sublimation of 2,5-DHB produced homogenous coatings yielding excellent PL signals in both positive and negative polarities. These high quality coatings allowed us to investigate molecular composition down to a spatial resolution of 5µm.
Sagittal whole body sections were cut from a 2-day old mouse pup measuring 1x3cm. PL distributions were surveyed over the sections with a spatial resolution of 200µm. Numerous organ specific signals were detected. For example, m/z 844.5 was highly abundant in the liver, whereas m/z 810.6 was unique to the adrenalin gland.
Coronal adult mouse brain sections were investigated with a range of resolutions. A first section was surveyed at 100µm to assess the PL expression of brain substructures. Numerous signals, such as m/z 826.7, were specific to the corpus callosum, while other signals such as m/z 734.7 were distributed within the cerebral cortex and striatum. Higher resolution data acquired at 25, 10 and 5µm demonstrated PL signals, including m/z 673.7 and 886.3, localized within striatum bundles.
PL distribution of pulmonary valve from adult mouse heart, a 10x600µm structure, was imaged at a 10µm resolution. Molecular composition was found to involve numerous ions, such as m/z 723.7 and 848.7.
These data demonstrate that matrix deposition via sublimation produces homogenous coatings that allow high resolution MALDI imaging MS down to the cellular level.
