Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity

Abstract

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 obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation stress. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical systems.

Origin of dispersive line shapes in plasmon-enhanced stimulated Raman scattering microscopy

“…The PESRS spectra were simulated by using a plasmon response with Δ = 5000 cm −1 , A = 100, and Ω R = 800 nm. Experimentally, the enhancement factor of PESRS signal is about 10 6 -10 7 in previous results [9,13]. The PESRS signal is proportional to the cubic of the plasmonic field, as shown in our theory.…”

“…We have experimentally investigated the line shapes of PESRL and PESRG predicted by our theoretical model above. The hyperspectral SRS data are collected by using a spectral-focusing SRS microscope which has been previously described [13]. Figure 3 presents the scheme of the hyperspectral SRS microscope.…”

“…Unlike the Lorentzian-line shape in traditional SRS spectroscopy, SE-FSRS spectroscopy and PESRS hyperspectral imaging both produced dispersive line shape spectra [9][10][11][12][13][14][15]. The dispersive line shape was attributed to Fano interference of the discrete molecular vibrational states on the continuum-like plasmon resonance [9,11].…”

“…The Van Duyne's group reported surfaceenhanced femtosecond SRS (SE-FSRS) spectra, including stimulated Raman loss (SRL) and stimulated Raman gain (SRG) from molecules embedded in a SiO 2 -coated gold nanodumbbell colloid [9][10][11][12]. Very recently, Cheng and coworkers demonstrated a label-free plasmon-enhanced SRS (PESRS) microscopy which is able to detect single molecules having extremely small Raman cross-sections (∼10 −30 cm 2 sr −1 ) with the aid of gold nanoparticles (Au NPs) and the pico-Joule high-repetition-rate laser pulses [13]. The reported PESRS microscopy opened a new window for ultrasensitive and ultrafast bio-imaging.…”

Nonlinear plasmon-exciton coupling enhances sum-frequency generation from a hybrid metal/semiconductor nanostructure

“…Compared to commonly adopted SH quantum channels, SF generation offers much wider spectral tuneability for the coherent manipulation of the linear and nonlinear fields, opening up new routes for optical control in such a nonlinearly coupled nanosystem. The results also opens up the possibility to explore difference frequency generation ω 1 À ω 2 j jof two plasmonic fields for coupling to low-frequency infrared or terahertz vibrations, for example, for plasmon-enhanced stimulated Raman scattering 44 .…”