Optical-Quality Assessment of a Miniaturized Intraocular Telescope.

Age-related macular degeneration (AMD) causes severe vision impairments, including blindness. An option to improve vision in AMD patients is through intraocular lenses and optics. Among others, implantable miniaturized telescopes, which direct light to healthy lateral regions of the retina, can be highly effective in improving vision in AMD patients. Yet, the quality of the restored vision might be sensitive to the optical transmission and aberrations of the telescope. To shed light on these points, we studied the in vitro optical performance of an implantable miniaturized telescope, namely, the SING IMT™ (Samsara Vision Ltd., Far Hills, NJ, USA) designed to improve vision in patients affected by late-stage AMD. Specifically, we measured the optical transmission in the spectral range 350-750 nm of the implantable telescope with a fiber-optic spectrometer. Wavefront aberrations were studied by measuring the wavefront of a laser beam after passing through the telescope and expanding the measured wavefront into a Zernike polynomial basis. Wavefront concavity indicated that the SING IMT™ behaves as a diverging lens with a focal length of -111 mm. The device exhibited even optical transmission in the whole visible spectrum and effective curvature suitable for retinal images magnification with negligible geometrical aberrations. Optical spectrometry and in vitro wavefront analysis provide evidence supporting the feasibility of miniaturized telescopes as high-quality optical elements and a favorable option for AMD visual impairment treatments.

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins.

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

Focus image scanning microscopy for sharp and gentle super-resolved microscopy.

To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.

Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping.

Three-dimensional imaging at high-spatiotemporal resolutions and over large penetration depths is key for unmasking the dynamics and structural organization of complex biological systems. However, the need to axially shift the focus, with consequent limitations in imaging speed, and signal degradation at large depths due to scattering effects, makes this task challenging. Here, we present a novel approach in 2-photon excitation microscopy that allows fast volumetric imaging and enhanced signal-to-background (S/B) in thick tissue. Our technique is based on ultrafast beam shaping at each pixel by means of an acoustic optofluidic lens. Shaping the excitation beam with different phase profiles enables both high-speed axial focus shifting, for continuous volumetric imaging, and controlled aberrated imaging, advantageous for out-of-focus background removal. We provide a theoretical description of our approach, and demonstrate volumetric imaging of neuronal cells from a mouse brain slice with enhancements in S/B up to a factor of 10 over a depth of 600 µm.

Direct Laser Printing of Tailored Polymeric Microlenses.

We report a laser-based approach for the fast fabrication of high-optical-quality polymeric microlenses and microlens arrays with controllable geometry and size. Our strategy consists of the direct laser printing of microdroplets of a highly viscous UV prepolymer at targeted positions, followed by photocuring. We study the morphological characteristics and imaging performance of the microlenses as a function of the substrate and laser parameters and investigate optimal printing conditions and printing mechanisms. We show that the microlens size and focusing properties can be easily tuned by the laser pulse energy, with minimum volumes below 20 fL and focal lengths ranging from 7 to 50 µm.