  Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free
chemical imaging of biomedical systems. The imaging sensitivity of SRS
microscopy is limited to ~10 mM for endogenous biomolecules. Electronic
pre-resonant SRS allows detection of sub-micromolar chromophores. However,
label-free SRS detection of single biomolecules having extremely small Raman
cross-sections (~10-30 cm2 sr-1) remains unreachable. Here, we demonstrate
plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with
single-molecule detection sensitivity. Incorporating pico-Joule laser
excitation, background subtraction, and a denoising algorithm, we obtained
robust single-pixel SRS spectra exhibiting the statistics of single-molecule
events. Single-molecule detection was verified by using two isotopologues of
adenine. We further demonstrated the capability of applying PESRS for
biological applications and utilized PESRS to map adenine released from
bacteria due to starvation stress. PESRS microscopy holds the promise for
ultrasensitive detection of molecular events in chemical and biomedical
systems.
