Dynamics of Oriented Arrangement of Gold Nanorods via Coherent Modulation Amplitude Projection

Gold nanorods (AuNRs) are highly promising biomedical probes due to their tunable plasmonic properties, yet real-time, high-resolution imaging of their subcellular behavior—particularly their orientation dynamics, which reflect critical nano-bio interactions—has been hindered by the diffraction limit and drawbacks of existing super-resolution methods, such as reliance on high-intensity lasers and exogenous labeling. To address this, we developed Coherent Modulation Amplitude Projection Imaging (CMAPI), a novel label-free technique that uses spatially and temporally modulated pairs of femtosecond pulses to coherently control the two-photon photoluminescence (TPPL) of AuNRs. By exploiting AuNRs as three-level systems with a measurable intermediate state, CMAPI encodes sub-diffraction-limit spatial and orientational information into the frequency domain through precise manipulation of inter-pulse delay, phase, and polarization. Experimental results confirmed the nonlinear excitation nature of AuNRs, with single-pulse polarization response following a cos²θ dependence. Under two-pulse excitation, the emission exhibited distinct coherence-dependent behavior: at zero delay, the response was governed by quantum superposition; at a delay matching the intermediate state lifetime (0.5 ps), a three-level model accurately described the response; and at longer delays (10 ps), the system reverted to incoherent emission. CMAPI retrieves nanoscale information via Fourier analysis of photon arrival times, producing simultaneous amplitude and phase images that reveal AuNRs’ precise positions (∼60 nm localization precision), in-plane orientations (e.g., quadrant-specific alignment inferred from phase sign), and local environmental coupling, such as plasmon-induced phase jumps, all under ultralow excitation power (<5 μW/μm²) to avoid photodamage. This approach enables visualization of features beyond the diffraction limit, distinguishing multiple AuNRs within a single diffractive spot, as validated by scanning electron microscopy. CMAPI provides a powerful, non-invasive platform for quantifying dynamic biological processes involving anisotropic nanoparticles, including conformational shifts during endocytosis, torque transmission in molecular motors, and real-time tracking of nanoscale interactions, thereby offering profound insights for theranostic probe design and fundamental biophysical research.