Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

Abstract

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon-deuterium bonds (C-D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.stable isotope labeling | stimulated Raman microscopy | protein synthesis

Raman spectroscopy: techniques and applications in the life sciences

“…Other features correspond to DNA and part of the higher-wavenumber water tail. Vibrations from Raman labels such as stable-isotope and particularly alkyne tags have bands in the lower 2000 cm −1 range, and have been utilised multiple times in SRS studies [251,252]. Initially lipids and proteins were most commonly measured with SRS in cells and tissues, as they contain the strongest and most intense contributions in the high-wavenumber region.…”

“…Initially lipids and proteins were most commonly measured with SRS in cells and tissues, as they contain the strongest and most intense contributions in the high-wavenumber region. Wei et al used SRS combined with various stable-isotope labeled amino acids, which when metabolically incorporated into cells was able to be used to image newly synthesised proteins [251]. Alkyne labels are also powerful for SRS imaging, and are discussed in Section 4.10 in more detail.…”

Ultrafast 2D IR microscopy

“…To take advantage of the high time resolution and dynamical information afforded by ultrafast IR spectroscopy, and inspired by fluorescence lifetime imaging microscopy (FLIM), a method widely used for biological imaging [27,28], we demonstrate the use of vibrational lifetime as a contrast agent for spatial mapping of heterogeneous chemical (solvation) environments. The rate of vibrational energy relaxation of a chromophore reports on the local solute-solvent interactions, serving as the contrast agent for imaging heterogeneous environments, such as solvent polarity or viscosity distributions.…”

Label-free real-time imaging of myelination in theXenopus laevistadpole byin vivostimulated Raman scattering microscopy

“…Therefore, SRS microscopy enables us to probe molecule's chemical bond in cells or tissues and provides us with information regarding their molecular composition. [32][33][34][35] As illustrated in Fig. 1(a), the pump field (ω p ) and the Stokes field (ω S ) excite the molecules from the ground state to the excited vibrational state by passing through a virtual state.…”