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.

Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.

A system of more than one part can be deciphered by observing differences between the parts. A simple way to do this is by recording something absolute displaying a trait in one part and not in another: in other words, mutually exclusive computation. Conditional directed expression in vivo offers processing in more than one part of the system giving increased computation power for biological systems analysis. Here, I report the consideration of these aspects in the development of an in vivo screening assay that appears sufficient to identify components specific to a system.