Biology subjects

Saliba, N.

Publications and source records attributed to Saliba, N..

4 recordsLinked to original sources

bioRxiv2026

High-speed volumetric single-molecule imaging using dual-wavelength light sheets and PSF-engineered enhanced biplane detection

Single-molecule localization microscopy (SMLM) enables nanoscale imaging but remains limited in three-dimensional (3D), high-speed, and high-density applications due to background fluorescence, photon inefficiency, and large point-spread function (PSF) footprints. Here, we present single-objective light-sheet microscopy with PSF-engineering enhanced biplane detection (SoLiD-3D), a versatile imaging platform that integrates dual-wavelength light-sheet illumination with dual-color, multi-configuration biplane imaging for parallel acquisition with PSF engineered detection for high-speed volumetric SMLM. Parallelized single-objective light-sheet excitation combined with PSF engineering overcomes key limitations of conventional wide-field and biplane approaches. Independent control of two excitation wavelengths for optical sectioning enables simultaneous dual-target imaging and single-target dual-color imaging with improved contrast and temporal resolution utilizing dynamically displaced light sheets for volumetric coverage. Using SoLiD-3D, we demonstrate high-speed single- and dual-target dual-color imaging that doubles localization density without sacrificing photon efficiency and continuous volumetric imaging via PSF-engineering enhanced biplane detection for whole-cell 3D imaging with improved axial localization performance over extended depth ranges. We further demonstrate improved speed by utilizing the Hummus PSF, a compact engineered PSF that enables high-precision 3D localization with a substantially reduced spatial footprint, for the first time for super-resolution imaging applications. Taken together, SoLiD-3D mitigates the trade-off between axial range and localization precision and offers improved speed compared to conventional 3D SMLM approaches.

biophysics↗

bioRxiv2026

Versatile and scalable reflective micromirrors for single-objective light sheet microscopy

We present a tunable microfabrication pipeline for creating robust, reflective inserts that adapt conventional commercial imaging chambers for single-objective light sheet (LS) illumination. This system reduces the complexity associated with dual-objective LS setups and specialized LS chambers while retaining the native functionality and biocompatibility of the original chambers. The fabricated insert features a metalized, 3D nanoprinted micromirror with an angled reflective surface, enabling alignment of a thin LS for sectioning and imaging throughout mammalian cells. Using this pipeline, we demonstrate that single-objective LS illumination achieves an over 4X improvement in the signal-to-background ratio compared with conventional widefield epi-illumination in both fixed and live cell samples. Furthermore, we show substantial resolution enhancement for single-molecule localization microscopy compared to epi-illumination for improved imaging at the nanoscale. The versatile and scalable design offers an easily implemented approach to bring the benefits of single-objective LS microscopy to a wide array of biological studies.

biophysics↗

bioRxiv2025

Single-objective lattice light sheet microscopy with microfluidics for single-molecule super-resolution imaging of mammalian cells

Single-molecule localization microscopy (SMLM) has redefined optical imaging by enabling imaging beyond the diffraction limit, allowing nanoscale investigation into cellular architecture and molecular dynamics. Light sheet illumination enhances SMLM through optical sectioning of the sample, which drastically improves the signal-to-background ratio and reduces photobleaching and photodamage. Lattice light sheet (LLS) microscopy, in which a 2D optical lattice is implemented for light sheet illumination, can provide exceptional sectioning and extended imaging depth when imaging in scattering samples. However, its conventional dual-objective design poses challenges for certain applications. Here, we present an imaging platform which implements LLS illumination with a reflective single-objective geometry (soLLS) inside a microfluidic chip, enabling the use of a single high numerical aperture objective for both illumination and detection, mitigating constraints of a dual-objective setup. We provide quantitative characterization of the propagation properties of the soLLS and demonstrate that it outperforms conventional Gaussian light sheets in terms of useful field of view and sectioning when propagating through scattering samples. Next, by combining soLLS with point spread function engineering, we demonstrate the platform for improved 3D single-molecule super-resolution imaging of multiple targets across multiple cells. The soLLS imaging platform thus expands investigations of nanoscale cellular and intercellular structures and mechanisms into more challenging samples for a wide range of applications in biology and biomedicine.

biophysics↗

bioRxiv2023

Whole-cell multi-target single-molecule super resolution imaging in 3D with microfluidics and a single-objective tilted light sheet

Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable novel microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. By combining these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets, we demonstrate whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.

biophysics↗