Probing Methionine Uptake in Live Cells by Deuterium Labeling and Stimulated Raman Scattering

Abstract

The small biomolecule methionine (Met) is a fundamental amino acid required for a vast range of biological processes such as protein synthesis, cancer metabolism, and epigenetics. However, it is still difficult to visualize the subcellular distribution of small biomolecules including Met in a minimally invasive manner. Here, we demonstrate stimulated Raman scattering (SRS) imaging of cellular uptake of deuterated methionine (d 8 -Met) in live HeLa cells by way of comparison to the previously used alkyne-labeled Met analoguehomopropargylglycine (Hpg). We show that the solutions of d 8 -Met and Hpg have similar SRS signal intensities. Furthermore, by careful image analysis with background subtraction, we succeed in the SRS imaging of cellular uptake of d 8 -Met with a much greater signal intensity than Hpg, possibly reflecting the increased and minimally invasive uptake kinetics of d 8 -Met compared with Hpg. We anticipate that d 8 -Met and other deuterated biomolecules will be useful for investigating metabolic processes with subcellular resolution.

Imaging the uptake of deuterated methionine in Drosophila with stimulated Raman scattering

“…The integrated SRS and fluorescence imaging system used in this study is described previously ( Shou et al, 2021 ; Spratt et al, 2022 ). The central wavelength of the pump pulses is fixed at 843.26 nm or 790 nm, with a spectral width of 0.15 nm for imaging the silent (2,000–2,300 cm −1 ) or CH stretching (2,800–3,100 cm −1 ) regions, respectively.…”

“…After confirming the position of the spectral peak for d 8 -Met, measurements were taken for four spectral points each with 1,500 frames: 2,107, 2,127, 2,130, and 2,150 cm −1 . SRS signal of d 8 -Met was obtained as , where corresponds to the SRS signal of the th spectral image ( Spratt et al, 2022 ). Lipid images with CH 2 stretching mode were taken at 2,853 cm −1 .…”

“…Mass spectrometry techniques ( Hu et al, 2020 ; Goncalves et al, 2022 ) provide detailed molecular information and can include spatial resolution ( Goncalves et al, 2022 ), but is destructive to cells and tissue, making it non-permissive for in situ testing. Click chemistry ( Bird et al, 2021 ; Scinto et al, 2021 ) has more recently been used to bypass the cumbersome nature of the traditional probes in high resolution fluorescence microscopy, but these probes may not be non-invasive ( Scinto et al, 2021 ; Spratt et al, 2022 ) and may not allow for the use of live cells. Additionally, most of these technologies have limited capabilities for long-term imaging.…”

“…Its metabolic product, S-adenosyl methionine (SAM), regulates stem cell division and differentiation ( Shiraki et al, 2014 ; Obata et al, 2018 ), and Met is essential for cancer cell division ( Hoffman and Erbe, 1976 ; Kaiser, 2020 ). In our recent work, we used deuterated methionine (d 8 -Met) and demonstrated the ability to detect the CD stretching peak of d 8 -Met by careful subtraction of background signal ( Spratt et al, 2022 ).…”

Unprecedented Regioselective Deuterium‐Incorporation of Alkyltrimethylammonium Chlorides and Raman Analysis

“…Therefore, Raman scattering of the deuterated compounds can help reveal interactions between the target natural materials and deuterated compounds (for example, interaction of the cell and deuterated unsaturated fatty acids [4a] ). [4] Because the weak sensitivity of C−D bonds is generally considered a problem in spontaneous Raman scattering, stimulated Raman scattering (SRS) has recently been developed to solve this problem [3a,b,4e] …”