Label-free 3D molecular imaging of living tissues using Raman spectral projection tomography

• Published in: Nature communications Vol. 15; no. 1; pp. 7717 - 11
• Main Authors: Stepula, Elzbieta, Walther, Anders R., Jensen, Magnus, Mehrotra, Dev R., Yuan, Mu H., Pedersen, Simon V., Kumar, Vishal, Gentleman, Eileen, Albro, Michael B., Hedegaard, Martin A. B., Bergholt, Mads S.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.09.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 13/107; 140/133; 631/1647/245/2221; 631/1647/527/1821; 639/624/1111/55; Animals; Blurring; Cartilage - diagnostic imaging; Cartilage - metabolism; Extracellular matrix; Extracellular Matrix - metabolism; Humanities and Social Sciences; Humans; Image contrast; Image reconstruction; Image resolution; Imaging, Three-Dimensional - methods; Information processing; Labels; Medical imaging; Mesoscale phenomena; Mice; Molecular Imaging - methods; multidisciplinary; Multivariate analysis; Phantoms, Imaging; Raman spectroscopy; Science; Science (multidisciplinary); Software; Spatial discrimination; Spatial resolution; Spectrum Analysis, Raman - methods; Tissue engineering; Tissue Engineering - methods; Tomography; Tomography - methods
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-51616-y

The ability to image tissues in three dimensions (3D) with label-free molecular contrast at the mesoscale would be a valuable capability in biology and biomedicine. Here, we introduce Raman spectral projection tomography (RSPT) for volumetric molecular imaging with optical sub-millimeter spatial resolution. We have developed a RSPT imaging instrument capable of providing 3D molecular contrast in transparent and semi-transparent samples. We also created a computational pipeline for multivariate reconstruction to extract label-free spatial molecular information from Raman projection data. Using these tools, we demonstrate imaging and visualization of phantoms of various complex shapes with label-free molecular contrast. Finally, we apply RSPT as a tool for imaging of molecular gradients and extracellular matrix heterogeneities in fixed and living tissue-engineered constructs and explanted native cartilage tissues. We show that there exists a favorable balance wherein employing Raman spectroscopy, with its advantages in live cell imaging and label-free molecular contrast, outweighs the reduction in imaging resolution and blurring caused by diffuse photon propagation. Thus, RSPT imaging opens new possibilities for label-free molecular monitoring of tissues. RSPT enables 3D imaging of tissues with label-free molecular contrast at mesoscale resolution. Here, the authors demonstrate its effectiveness in visualizing complex phantoms and tissue heterogeneities.