Bioorthogonal chemical imaging of metabolic activities in live mammalian hippocampal tissues with stimulated Raman scattering

Abstract

Brain is an immensely complex system displaying dynamic and heterogeneous metabolic activities. Visualizing cellular metabolism of nucleic acids, proteins, and lipids in brain with chemical specificity has been a long-standing challenge. Recent development in metabolic labeling of small biomolecules allows the study of these metabolisms at the global level. However, these techniques generally require nonphysiological sample preparation for either destructive mass spectrometry imaging or secondary labeling with relatively bulky fluorescent labels. In this study, we have demonstrated bioorthogonal chemical imaging of DNA, RNA, protein and lipid metabolism in live rat brain hippocampal tissues by coupling stimulated Raman scattering microscopy with integrated deuterium and alkyne labeling. Heterogeneous metabolic incorporations for different molecular species and neurogenesis with newly-incorporated DNA were observed in the dentate gyrus of hippocampus at the single cell level. We further applied this platform to study metabolic responses to traumatic brain injury in hippocampal slice cultures, and observed marked upregulation of protein and lipid metabolism particularly in the hilus region of the hippocampus within days of mechanical injury. Thus, our method paves the way for the study of complex metabolic profiles in live brain tissue under both physiological and pathological conditions with single-cell resolution and minimal perturbation.

Applications of vibrational tags in biological imaging by Raman microscopy

“…Later Chen et al created different vibrational colors for EdU by isotope-editing the alkyne moiety 21 (Figure 1E–F). With these multi-color labels and applying them in a pulse-chase manner, Hu et al recently showed that even different stages of DNA synthesis could be visualized in the same nuclei of neural progenitor cell 22 (Figure 1G–I). Thus, the live-cell imaging capability of SRS as well as the multiplexing potential of EdU makes it possible to reveal cell cycle dynamics in complex tissue or animal samples.…”

“…With all the tools developed for imaging nucleic acid, amino acids, fatty acid, and choline by SRS as previously described, Hu et al further studied complex metabolism with all these vibrational tags in live rat brain hippocampal tissues and the metabolic response after traumatic injury with subcellular resolution 22 . In the dentate gyrus of hippocampus, they found heterogeneous incorporations of amino acids, choline and fatty acids at subcellular level.…”

“…(E and F) structures and Raman spectra of isotope-edited EdU molecules 21 . (G to I) SRS imaging of 12 C-EdU (G) 13 C-EdU (H) channels and their overlay (I) in a pulse chase labeled neuron progenitor cell 22 . Scale bar: 20 μm.…”

“…Scale bar: 10 μm. (E–F) Increased choline (E) and fatty acid (F) metabolism for lipid synthesis are observed in the hilus of dentate gyrus after mechanical stretch 22 . Scale bars: 40 μm.…”

Optical Imaging of Metabolic Dynamics in Animals

“…Previously, our lab and others have developed methods to visualize lipogenic activities in living tissues by supplying deuterium-labeled fatty acids (D-FAs), such as palmitic acid, oleic acid, and arachidonic acid, and imaging C-D bonds in newly synthesized lipids [30][31][32][33] . However, DO-SRS is fundamentally different from those previous methods because D2O is a noninterfering probe that does not change native metabolism and is a non-carbon tracer that can probe activities of de novo lipogenesis.…”