Abstract
Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. Multimodal combinations of LSFM with additional analyses of the identical sample help to limit the consumption of restricted specimen and reduce inter-sample variation. Here, we demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Samples of freshly dissected murine brain and of archived formalin-fixed paraffin-embedded (FFPE) human brain tissue were cleared (3DISCO). Tissue regions of interest were defined by LSFM and excised, (re)-embedded in paraffin, and sectioned. Mouse sections were coated with sinapinic acid matrix. Human brain sections were pre-digested with trypsin and coated with α-cyano-4-hydroxycinnamic acid matrix. Subsequently, sections were subjected to MALDI-time-of-flight (TOF)-MSI in mass ranges between 0.8 to 4 kDa (human tissue sections), or 2.5–25 kDa (mouse tissue sections) with a lateral resolution of 50 µm. Protein- and peptide-identities corresponding to acquired MALDI-MSI spectra were confirmed by parallel liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The spatial abundance- and intensity-patterns of established marker proteins detected by MALDI-MSI were also confirmed by immunohistochemistry.
Highlights
Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs
Light sheet fluorescence microscopy (LSFM) of optically cleared biological tissue samples has rapidly evolved during the last decade and established as a powerful tool for 3-dimensional analysis of tissue morphology applied in various life science d isciplines[3,4,5]
Additional tissue sections were subsequently cut from the same block and processed for proteomic analysis by liquid chromatography-tandem mass spectrometry (LC–mass spectrometer (MS)/MS)
Summary
Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. We demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/ Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Combined 3D-imaging- and subsequent advanced analysis approaches allow for a precise definition of the tissue localizations to be further analyzed, and enable a direct morphological co-localization of the results of different downstream analyses types and their integration into superordinate functional concepts. Multimodal combination of LSFM with additional tissue-based morphological analyses is beneficial, since it enables the integration of the results of different analysis methods into the superordinate 3D morphology of the examined sample. We asked, if LSFM of optically cleared tissue samples would be combinable with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI). The solvent extracts the analyte molecules from the section followed by matrix/analyte co-crystallization preserving analytes position to their origin in the tissue section beneath
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
