Visualization of Cataract Surgery Steps With 4D Microscope-Integrated Swept-Source Optical Coherence Tomography in Ex Vivo Porcine Eyes.

Purpose: To investigate the visualization capabilities of high-speed swept-source optical coherence tomography (SS-OCT) in cataract surgery. Methods: Cataract surgery was simulated in wet labs with ex vivo porcine eyes. Each phase of the surgery was visualized with a novel surgical microscope-integrated SS-OCT with a variable imaging speed of over 1 million A-scans per second. It was designed to provide four-dimensional (4D) live-volumetric videos, live B-scans, and volume capture scans. Results: Four-dimensional videos, B-scans, and volume capture scans of corneal incision, ophthalmic viscosurgical device injection, capsulorrhexis, phacoemulsification, intraocular lens (IOL) injection, and position of unfolded IOL in the capsular bag were recorded. The flexibility of the SS-OCT system allowed us to tailor the scanning parameters to meet the specific demands of dynamic surgical steps and static pauses. The entire length of the eye was recorded in a single scan, and unfolding of the IOL was visualized dynamically. Conclusions: The presented novel visualization method for fast ophthalmic surgical microscope-integrated intraoperative OCT imaging in cataract surgery allowed the visualization of all major steps of the procedure by achieving large imaging depths covering the entire eye and high acquisition speeds enabling live volumetric 4D-OCT imaging. This promising technology may become an integral part of routine and advanced robotic-assisted cataract surgery in the future. Translational Relevance: We demonstrate the visualization capabilities of a cutting edge swept-source OCT system integrated into an ophthalmic surgical microscope during cataract surgery.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.