Depth-enhanced molecular imaging with two-photon oblique plane microscopy

High-numerical-aperture (NA) oblique plane microscopy enables noninvasive fluorescence imaging of subcellular dynamics without radical sample modification. However, performance degrades at depth in multicellular specimens as scattering and refractive-index heterogeneity raise out-of-focus background. We report a two-photon oblique plane microscope that improves resolution at depth by combining high-NA single-objective detection with multiphoton plane illumination. The microscope achieves ~ 300 nm lateral and ~ 650 nm axial resolution, with single-molecule sensitivity and < 60 nm localization precision in vivo. Compared with two-photon point scanning, the lower illumination NA delivers order-of-magnitude lower peak intensity, enabling > 5 × faster volumetric acquisition (up to 3.25 × 10 6 voxels s −1 ). In multicellular contexts, near-infrared nonlinear excitation enhances contrast throughout the illumination depth by ~ 2 × and restores volumetric resolving power by > 2 × relative to linear excitation. We demonstrate these capabilities through molecular imaging of epithelial tissue, stem-cell-derived gastruloids, and living fruit fly embryos, including multicolor transcription-factor dynamics, optogenetic subcellular control, and single-mRNA tracking, all using standard glass-based mounting.