Significance: Integrating ultrasound (US) with multiphoton microscopy (MPM) enables comprehensive tissue characterization by combining contrast mechanisms across spatial scales. However, prior implementations suffer from degraded US image quality at the glass optical window arising from off-axis acoustic noise (clutter).
Aim: We aimed to reduce clutter near the optical window without compromising the MPM image quality using different optical window materials combined with different ultrasound beamforming techniques.
Approach: Clutter in B-mode images was measured for glass and several optically clear polymer films using physically or synthetically focused US beamforming with varied focal configurations ( ) and with acoustic coupling beneath the window. MPM image quality with the material optical windows was assessed via second harmonic generation (SHG) point spread function (PSF) measurements. We evaluated SHG-based automated collagen fiber alignment quantification with the optical windows in rat tail tendon samples and collagen gels.
Results: Candidate materials included polymethyl pentene (PMP), cyclin olefin copolymer (COC), polycarbonate (PC), polyethylene terephthalate, polymethyl methacrylate, and an Ibidi® polymer coverslip. At receive , COC and Ibidi® reduced acoustic clutter by compared with glass with physical focusing regardless of physically or synthetically focused beams or acoustic coupling. Collagen gel imaged with COC and high showed clearer ultrasound speckle close to the optical windows. COC, Ibidi®, PMP, and PC showed minimal SHG PSF differences from glass and collagen alignment errors relative to glass.
Conclusions: COC or Ibidi® optical windows significantly improve US image quality without compromising MPM quality. These enhancements support future multimodal studies with high-quality, co-registered US and MPM data.
Keywords: acoustic clutter; collagen; multimodal; multiscale; nonlinear optics; second-harmonic generation; ultrasound.
© 2025 The Authors.