Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

Abstract

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans.

Applications of vibrational tags in biological imaging by Raman microscopy

“…Wei et al further improved the label incorporation using a mixture of 20 deuterated amino acids, and applied SRS for fast image acquisition 28 (Figure 2B–E). This methodology is later optimized into a general platform to interrogate protein metabolism across a variety of systems, including primary neuron cells, brain tissues, and even whole animals including zebrafish larvae and mouse 29 (Figure 2F and G). They revealed possibly function-related higher protein synthesis activity in hippocampus and cortical regions in a brain slice.…”

“…By targeting carbon hydrogen bonds and carbon deuterium bonds respectively, they achieved ratiometric images between existing proteins and newly synthesized proteins, and identified newly grown neurites which had a higher percentage of newly synthesized proteins (Figure 5A). And in the follow-up study by the same group, where all 20 amino acids were replaced by their deuterium substituted counterparts in the custom-prepared media, the protein synthesis on an entire ex vivo organotypic brain slice was quantitatively visualized 29 . (Figure 5B) The authors found that the protein synthesis was only active in the dentate gyrus of the hippocampal region and in a few individual neurons.…”

“…(F and G) SRS imaging of newly synthesized proteins in liver. (F) and intestine (G) tissue sections of mouse after administration of deuterated amino acid 29 . Scale bar: 10 μm.…”

Coherent Raman Scattering Microscopy in Biology and Medicine

“…Using SRS microscopy, Zhang et al (42) studied cellular lipid uptake using treatment with deuterated fatty acids. Deuterated compounds have also helped visualize important metabolites in cells, such as protein and choline (112, 116, 117). Shen et al (115) studied protein degradation in cells with carbon-isotope-labeled SRS imaging.…”

D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

“…Deuterium-based Raman labels have been successfully used in nonlinear optical imaging applications to help increase specificity to water dynamics, 21 selected phospholipids, 22,23 neutral lipids, [24][25][26] and protein metabolism. 27,28 Labels based on stable isotopes, including deuterium, have found widespread use in clinical applications. Because the level of toxicity of pharmacological substances and their deuterated analogs is typically identical, deuterated substances are widely used in clinical studies aimed at studying the metabolism and movement of drugs in humans.…”