Multimodal Spectral Microscopy And Imaging Mass Spectrometry Of Biomolecules In Cells And Tissues

Biomolecular imaging enables localization and characterization of biomolecules at the cellular and tissue levels in order to better understand their roles in biological organisms. Although a plethora of imaging techniques is available, there is a need to improve current imaging methodologies and apply them for selective analyses of complex biological specimens. The work described in this thesis is related to development and applications of spectral imaging and imaging mass spectrometry (IMS) methodologies. Spectral imaging uses either labeling dyes or native chromophores to visualize biomolecules in cells and tissues under a specialized light microscope, while IMS provides direct detection and analysis of biomolecules on tissue surfaces with preserved spatial distribution. In this initial study, multimodal spectral imaging of cells, cellular organelles, and tissues was performed on a light microscope upon the addition of a transmission diffraction grating. The instrument concurrently recorded spectral images by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and were imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield spectral microscopy, respectively. In the second project, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was utilized to visualize the protein spatial distribution on tissue surfaces. Our study has been focused on imaging of proteins in kidney tissue sections originating from apparently healthy (normal) mice and mice with polycystic kidney disease (PKD). Reconstruction of protein ion maps and identification of proteins by on-tissue enzymatic digestion were performed. MALDI-MS images were correlated with histological features obtained by hematoxylin and eosin staining, providing localization of peptides in different parts of the kidney (renal pelvis, medulla, and cortex). Nano-HPLC was coupled with imaging mass spectrometry to identify proteins in kidney tissue sections. Statistical analyses such as principle component analysis (PCA) was employed to achieve tissue classification and differentiate between healthy and diseased tissues. Experiments are underway to discover and validate proteins that can become potential biomarkers of PKD. In summary, we demonstrated that fluorescence, scattering, and absorption spectral images of cells can be acquired by incorporation of a transmission diffraction grating into a light microscope. The present spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens. A reproducible MALDI-IMS workflow was established and resulted in protein mass distribution maps of normal and PKD kidney tissues. Statistical comparison of MALDI-MS images provides classification of healthy and diseased tissues. Several proteins were localized in tissues and their identity will be further confirmed by HPLC combined with tandem mass spectrometry (MS/MS).