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

Abstract

Conspectus 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 that 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 the 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/deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and to 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 xenograft 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 fluorophore has done for larger molecular species.

Applications of vibrational tags in biological imaging by Raman microscopy

“…7–9 The very nature of label-free imaging techniques permits such applications to be performed in vivo . Chemical bonds such as O–P–O, C═O, C═C, S═O, O–H, C–H are frequently probed, especially C–H as it is highly enriched in lipids and proteins – the most abundant biomolecules in the cell – and thus gives the highest signal.…”

Electronic Preresonance Stimulated Raman Scattering Microscopy

“…44,45 Such bio-orthogonal chemical imaging offers a powerful platform for functional metabolic imaging in live cells and animals. 46−55 Its success underscores the importance of introducing vibrational probes to improve specificity and sensitivity of nonlinear Raman microscopy. However, even with extensive efforts of instrumentation improvement and small-tag optimization, the detection sensitivity of SRS is still in the range of 35 μM (i.e., diyne tags, double-conjugated alkynes) 51 to 200 μM (small alkyne tags).…”

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

“…As an emerging multiphoton optical imaging technique, stimulated Raman scattering (SRS) microscopy is sensitive and specific in detecting chemical bonds, offering diffraction-limited subcellular resolution, linear concentration dependence with background-free chemical contrast for quantitative measurement and intrinsic 3D optical sectioning capability1516171819202122. The use of picosecond excitation pulses and near-infrared wavelength also reduce photon scattering inside tissue samples and potential phototoxicity232425.…”