Advanced Registration and Analysis of MALDI Imaging Mass Spectrometry Measurements through Autofluorescence Microscopy

The
correlation of imaging mass spectrometry (IMS) with histopathology
can help relate novel molecular findings obtained through IMS to the
well-characterized and validated histopathology knowledge base. The
quality of correlation between these two modalities is limited by
the quality of the spatial mapping that is obtained by registration
of the two image types. In this work, we develop novel workflows for
MALDI IMS-to-microscopy data registration and analysis using nondestructive
IMS-compatible wide field autofluorescence (AF) microscopy combined
with computational image registration. First, a substantially automated
procedure for high-accuracy registration between IMS and microscopy
data of the same section is described that explicitly links the MALDI
laser ablation pattern imaged by microscopy to its corresponding IMS
pixel. Subsequent examination of the registered data allows for high-confidence
colocalization of image features between the two modalities, down
to single-cell scales within tissue. Building on this IMS-microscopy
spatial mapping, we furthermore demonstrate the automated spatial
correlation between IMS measurements from serial sections. This AF-registration-driven
inter-section analysis, using a combination of nonlinear AF-to-AF
and IMS-to-AF image registrations, can be applied to tissue sections
that are prepared and imaged with different sample preparations (e.g.,
lipids vs proteins) and/or that are measured using different spatial
resolutions. Importantly, all registrations, whether within a single
section or across serial sections, are entirely independent of the
IMS intensity signal content and thus unbiased by it.