Live-Cell Bioorthogonal Chemical Imaging: Stimulated Raman Scattering Microscopy of Vibrational Probes

Abstract

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, a bioorthogonal chemical imaging platform has recently been introduced. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes and stable isotopes), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides and deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". As a unique tool for biological discovery, this platform has been applied to studying various metabolic processes under both physiological and pathological states, including protein synthesis activity of neuronal systems, protein aggregations in Huntington disease models, glucose uptake in tumor xenografts, and drug penetration through skin tissues. We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species.

Figure. 1

Physical principle and instrumental setup for stimulated Raman scattering microscopy. (a) Energy diagrams for spontaneous Raman scattering, coherent Anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS). (b) Experimental setup of typical SRS microscopy. (c) A Raman spectrum of mammalian cell samples designating the crowded fingerprint region and the cell-silent region.

Figure. 2

Sensitive and specific SRS imaging for diverse alkyne-bearing small biomolecules. (a) Chemical structures of alkyne-tagged small biomolecules. (b-c) The corresponding spontaneous Raman spectra (b) and the SRS images (c) of each alkyne-tagged small biomolecules after metabolic incorporation into mammalian cells or neurons. Adapted from refs. and , copyrights 2014 Nature Publishing Group and 2015 John Wiley & Sons, Inc. Scale bar: 10 μm.

Figure. 3

SRS imaging of in vivo delivery of an alkyne-bearing small-molecule drug, Terbinafine Hydrochloride (TH) into mouse ear. (a) Spontaneous Raman spectrum of TH. (b) SRS images at selected depths for TH at the alkyne channel and for proteins and lipids at respective label-free amide and lipid channels in mouse ear tissues. Adapted from ref. , copyright 2014 Nature Publishing Group. Scale bar: 20 μm.

Figure. 4

SRS imaging of new protein synthesis by metabolic incorporation of deuterated amino acids (D-AAs). a) A cartoon illustrating the metabolic enrichment of D-AAs into cells’ nascent proteins. b) SRS images of newly synthesized proteins in live neurons, brain slices and animals by targeting the C-D vibrational peak. Adapted from Ref. , copyright 2015 American Chemical Society. Scale bar: 10 μm.

Figure. 5

SRS imaging of quantitative proteome degradation by metabolical incorporation of ¹³C-Phe. Time-dependent images at both ¹²C-Phe and ¹³C-Phe channels in live HeLa cells are obtained and the ratio maps of ¹²C/(¹²C+¹³C) show quantitative decay of the pre-existing proteome. Adapted from Ref. , copyright 2015 John Wiley & Sons, Inc. Scale bar: 20 μm.

Figure. 6

Bioorthogonal Chemical Imaging for the dynamic metabolism of small biomolecules. (a-b) Cell division tracking after incorporated with EdU for newly synthesized DNA (a) and D-AAs for newly synthesized proteins (b). (a) is adapted from ref. , copyright 2014 Nature Publishing Group. (b) is adapted from Ref. , copyright 2013 National Academy of Sciences. (c) Time-lapse imaging from 10 min to 5 h for protein synthesis with metabolic labeling of D-AAs. Adapted from Ref. , copyright 2015 American Chemical Society. Scale bar: 10 μm.

Figure. 7

Multicolor Bioorthogonal Chemical Imaging. (a) Multicolor SRS imaging of isotope-edited (EdU-¹³C, EU-¹³C₂) and unedited (17-ODYA) alkyne-tagged small biomolecules after metabolic incorporation into new DNA (EdU-¹³C), RNA (EU-¹³C₂), and triglycerides (17-ODYA). Adapted from Ref. , copyright 2015 American Chemical Society. (b) Sub-grouping of D-AAs with similar chemical environments. (c) Two-color pulse-chase imaging for the protein aggregation formation of mutant Huntingtin proteins by metabolic labeling of two groups of D-AAs. (b-c) adapted from Ref. , copyright 2015 American Chemical Society. Scale bar: 10 μm.