Fig.1. Imaging protein synthesis with metabolic incorporation of deuterium-labeled amino acids. By feeding live cells with deuterium-labeled amino acids, newly synthesized proteins can be specifically labeled with C-D bonds. By tuning the energy difference between Pump and Stokes beams to match the vibrational frequency of C-D, the distribution of C-D carrying new proteins can be imaged in live cells by SRS with high sensitivity and resolution without fixation or staining.
Research-SRS imaging of isotope tags
(1) Imaging protein synthesis in living cells and animals via C-D vibrations.
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 (e.g., learning and long-term memory) and pathological (e.g., Alzheimer’s disease and autism) conditions. However, selective visualization of a newly synthesized proteome in living systems with sub-cellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography and mass spectrometry.
We reported a novel imaging technique to visualize nascent proteins by harnessing the emerging SRS microscopy coupled with metabolic labeling of deuterated amino acids. Incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, while SRS imaging of exogenous C-D bonds in the cell-silent Raman region is highly sensitive, specific and compatible with living systems. Newly synthesized proteins are then imaged via their unique vibrational signature of C-D bonds (Fig. 1). Thus our technique of nonlinear vibrational imaging of deuterium incorporation will be a valuable tool to unravel the complex spatial-temporal dynamics of newly synthesized proteome in vivo.
As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining (Fig. 2). Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells can be clearly identified via this new method.
Fig.2. SRS imaging of newly synthesized proteins in live HeLa cells. (a) Raman spectrum of HeLa cells incubated with a medium containing deuterium-labeled amino acids for 20 hrs. (b) SRS image targeting the 2133 cm-1 peak of C-D shows a high-contrast image representing newly synthesized proteins. (c) SRS image of the same cells as in (b) at off-resonant 2000 cm-1. (d-f) SRS images of same cells at 1655 cm-1 (amide I); 2845 cm-1 (CH2) and 2940 cm-1 (CH3) show the intrinsic distributions of total cellular lipids and proteins.
Very recently, we have significantly improved the technique, such as maximizing the protein C-D labeling efficiency, and creating two-color pulse-chase imaging by separating essential amino acids into two distinct sets. For the applications, we have achieved imaging protein synthesis in live zebrafish larvae and organotypic mouse brain tissue slices. Interestingly, only a subpopulation of neurons in the dentate gyrus region is found to exhibit high synthesis activity (Fig. 3). For future works, we are planning to study the intricate relation between neuronal activity and local protein synthesis during learning and long-term memory.
Fig.3. SRS imaging of newly synthesized proteins in an acute brain tissue slice (the hippocampus region) from a wild type mouse. SRS image (green) of C-D shows newly synthesized proteins highly enriched in the dentate gyrus region. The 2845 cm-1 (CH2) channel shows the distributions of total lipids (purple), while 2920 cm-1 (CH3) channel shows the distributions of total proteins (red).
(2) Imaging proteins turnover via 13C incorporation.
Protein turnover, consisting both protein synthesis and degradation, is an indispensable regulatory process throughout cell division, growth, differentiation and diseases. Therefore it is highly desirable to develop a non-invasive live cell imaging technique to generate quantification map of protein turnover dynamics with subcellular resolution. We have developed a strategy that utilizes metabolic labeling of 13C stable isotopes to visualize both the original and the nascent proteome in a time dependent manner. We chose the strong ring breathing mode of (12C- or 13C-) phenylalanine as the spectroscopic marker of (old and new) proteome and quantified proteome turnover by ratio maps, as shown in Fig. 4.
We demonstrated the general applicability of our technique in revealing steady-state protein turnover in mammalian cell lines and single cell organisms (S. pombe yeast), and extended our efforts to image neurite outgrowth during PC12 cells differentiation and protein aggregation related to neurodegenerative diseases. To our knowledge, this is the first time that 13C labeled molecular species are probed by nonlinear vibrational microscopy, and protein degradation dynamics is directly imaged in live cells.
Fig.4. Proteome turnover of HeLa cells by SRS imaging. (a) Time dependent Raman spectra of HeLa cells incubated with 0.8 mM 13C-phenylalanine, showing a decline of 1004 cm-1 (12C) phenylalanine peak and a concurrent rise of 968 cm-1 phenylalanine (13C) peak. (b) Single exponential fit of 12C ratio calculated as 12C/(12C+13C) from spectra reveals a time constant of 43 hrs. (c) SRS images of HeLa cells. 13 ratio images depict a map of protein turnover. (d) Single exponential fit of 12C ratio from SRS images reveals a time constant of 40 hrs.
1. F. Hu, L. Wei, C. Zheng, Y. Shen and W. Min. "Vibrational imaging of choline metabolites in live cells by stimulated Raman scattering coupled with isotope-based metabolic labeling", Analyst (in press) 2014.
2. L. Wei, Y. Yu, Y. Shen, M. C. Wang and W. Min. "Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy", Proc. Natl. Acad. Sci. USA, 110, 11226, 2013.[PDF]