Deep and Dynamic Imaging

Overview

Metabolic and structural imaging of the entire 500-μm-deep living blood-brain barrier microfluidic model, comprising vascular endothelial cells, pericytes, and astrocytes.

To achieve deep-tissue imaging in living organisms, two-photon autofluorescence (2PAF) microscopy of NAD(P)H is a powerful technique, offering non-invasive, high-resolution visualization of cellular metabolic processes. However, light scattering traditionally limits the penetration depth of this method to within 200 μm. We have overcome this limitation by developing a high-power, multimode fiber-based light source that modulates multimodal nonlinear pulse propagation with a compact fiber shaper. This innovative approach extends the imaging depth of 2PAF microscopy to over twice its previous limit. The modular design provides flexibility and facilitates the widespread adoption of this technology for demanding in vivo and in vitro imaging applications, including in areas such as cancer research, immune responses, and tissue engineering.

Highlights

Source: A multimode fiber source generating 0.5 MW peak power at 1100±25 nm was achieved by adaptively modulating multimodal nonlinear pulse propagation with a compact fiber shaper.
Imaging: Three-photon excitation at 1100 nm enabled structural and metabolic imaging at depths exceeding 700 μm in living engineered human multicellular microtissues.
Biology: Revealed depth-associated redox heterogeneity in the blood-brain barrier microfluidic model and speed-associated redox heterogeneity in monocyte behaviors.

Concept of the imaging platform using multimode fiber source.

Results

Beam optimization for high-quality imaging

Comparison of images and beam properties acquired before (initial) and after (optimized) optimizing the fiber shaper.

Three-photon NAD(P)H imaging improves depth

Deep NAD(P)H imaging with 1100 nm MMF source through the entire 720 μm of depth of the 3D microvascular network. Three-photon imaging uses longer excitation wavelength that reduces the scattering in tissue. The higher-order confinement improves the imaging signal-to-background ratio (SBR) deep in tissue.

Deep imaging in blood-brain barrier microfluidic model

Deep metabolic and structural imaging of the entire 500-μm-deep living blood-brain barrier microfluidic model, comprising vascular endothelial cells, pericytes, and astrocytes. Based on the structural and metabolic features, same type of cells can cluster. Additionally, the ability to image deep into tissue reveals depth-associated redox heterogeneity that can help to understand biology.

Dynamic imaging of monocyte behaviors

Dynamic metabolic and structural imaging of the monocyte behaviors in the vasculature network, showing relation between metabolic and motility behaviors in individual cells as well as statistics in a large population.

Multimode fibers (MMFs) are gaining renewed interest for nonlinear effects due to their high-dimensional spatiotemporal nonlinear dynamics and scalability for high power. High-brightness MMF sources with effective control of the nonlinear processes would offer possibilities in many areas from high-power fiber lasers, to bioimaging and chemical sensing, and to intriguing physics phenomena. Here we present a simple yet effective way of controlling nonlinear effects at high peak power levels. This is achieved by leveraging not only the spatial but also the temporal degrees of freedom during multimodal nonlinear pulse propagation in step-index MMFs, using a programmable fiber shaper that introduces time-dependent disorders. We achieve high tunability in MMF output fields, resulting in a broadband high-peak-power source. Its potential as a nonlinear imaging source is further demonstrated through widely tunable two-photon and three-photon microscopy. These demonstrations provide possibilities for technology advances in nonlinear optics, bioimaging, spectroscopy, optical computing, and material processing.