Fig. 1. UMAMI system. (a) System schematic: a ∼2.1MHz annular US transducer is attached to the objective’s front aperture of a conventional TPM system (left) via a 3D printed holder (right) that co-registers the optical and US focal planes. (b) Expanded view of the relative size of the US (purple/gray) and optical (red) foci. Illustration of the pressure wave modulation during a line scan (right), highlighting how temporal variation in US pressure results in spatially patterned AOM.

Fig. 2. Giant AOM effect and its characteristics. (a) AOM in an agar-embedded bead cluster (top, left). The 2.1 MHz US wave is slowly gated on (red) and off (green) within the frame to highlight the modulation effect. The modulation of fluorescence from a representative line (red) as compared to the unmodulated baseline (green) demonstrated large fractional modulation (top, right). An in vivo AOM image and the modulation of fluorescence of a single line are shown as well (bottom). (b) Signal frequencies 0 to 5 MHz from an AOM in vivo frame. While the US is turned off, the image frequencies are predominantly contained around the DC; however, with the US on, large peaks at the US fundamental frequency and second harmonic are observed. (c), (d) The modulation of the fluorescence signal at the US fundamental frequency of 2.1 MHz is proportional to the US pressure’s amplitude (top, left) and independent of laser excitation intensity (top, right). (Seven beads, 24 frames averaged each.) (e), (f) Similar results obtained in vivo from (eGFP) labeled neurons 200μm from the brain surface. The blue dashed lines represent linear fits, while the green dots show respective baseline (US off) values.

Fig. 3. Demodulation for UMAMI imaging. (a) Computational strategy for image demodulation. A series of modulated images are motion corrected, transformed to the Fourier domain, and band-pass filtered around the fundamental US frequency (see Appendix). Band-passed signals are shifted to the low-frequency spectral range and inversely transformed back to the image domain. In step 2, demodulated frames are then averaged as needed until sufficient SNR is reached (right), providing additional information to the baseline (unfiltered) images. (b) Example demodulation process of a fluorescent bead embedded in agar and a non-fluorescent bead embedded in fluorescein-laden agar (left and right, respectively) (24 frames averaged). Original images are unfiltered and have no AOM (top). Demodulated images from when the US is on show pronounced signals at the edges of the beads (middle). When the US is turned off and images are demodulated, no signal is observed (bottom).

Fig. 4. UMAMI mechanism – conceptual model and simulation. (a) Comparison of a scan’s position versus time for a normal raster scan and a sinusoidally displaced UMAMI scan. (b) Schematic depiction of the origin of UMAMI generated fluorescent signals/fringe pattern. During a single line scan, fluorescent signals near the bead’s edge are sinusoidally modulated due to the movement across the PSF’s boundaries (points B and C) while center loci remain relatively constant during spatial deflection and have no resulting signal modulations (point A). (c) Top, the profile of a simulated bead under a regular raster scan versus UMAMI scan. Middle, the band-passed UMAMI scan shows that the modulation amplitude is largest at the edges, where the fluorescent derivatives are greatest, and minimal at the center of the bead. Inset: spectral content of the band-passed modulation signal. Bottom, the results of applying our demodulation procedure and a bandstop filter on the simulated UMAMI scanned signal showing enhanced edges as well as recovery of the original profile. (d) Theoretical dependence of the modulation ratio on the displacement amplitude. The displacement amplitude is putatively proportional to US pressure, leading to an overall linear dependence on pressure.

Fig. 5. UMAMI of cortical neurons in vivo reveals enhanced detail and contrast. (a) Demonstration of improved optical sectioning in densely labeled (GCaMP) cortical layer 2/3 neurons (left). Z-stacks for baseline and during AOM. Right: the higher zoom stack highlights rejection of out-of-focus fluorescence, revealing the nuclear exclusion of eGFP. (b) Baseline and demodulated neuron images: green (baseline) and red (AOM demodulated) lines show enhanced contrast, as well as background rejection, revealing nuclear fluorescent protein exclusion.