arXiv subjects

Firehun Tsige Dullo

Publications and source records attributed to Firehun Tsige Dullo.

2 recordsLinked to original sources

arXiv2020

A transparent waveguide chip for versatile TIRF-based microscopy and nanoscopy

Total internal reflection fluorescence microscopy (TIRF) has enabled low-background, live-cell friendly imaging of cell surfaces and other thin samples thanks to the shallow penetration of the evanescent light field into the sample. The implementation of TIRF on optical waveguide chips (c-TIRF) has overcome historical limitations on the magnification and field of view (FOV) compared to lens-based TIRF, and further allows the light to be guided in complicated patterns that can be used for advanced imaging techniques or selective stimulation of the sample. However, the opacity of the chips themselves has thus far precluded their use on inverted microscopes and complicated sample preparation and handling. In this work, we introduce a new platform for c-TIRF imaging based on a transparent substrate, which is fully compatible with sample handling and imaging procedures commonly used with a standard #1.5 glass coverslip, and is fabricated using standard complementary metal-oxide-semiconductor (CMOS) techniques, which can easily be scaled up for mass production. We demonstrate its performance on synthetic and biological samples using both upright and inverted microscopes, and show how it can be extended to super-resolution applications, achieving a resolution of 116 nm using super resolution radial fluctuations (SRRF). These new chips retain the scalable FOV of opaque chip-based TIRF and the high axial resolution of TIRF, and have the versatility to be used with many different objective lenses, microscopy methods, and handling techniques. We thus see c-TIRF as a technology primed for widespread adoption, increasing both TIRF's accessibility to users and the range of applications that can benefit from it.

physics.app-ph↗

arXiv2019

Photonic-chip assisted correlative light and electron microscopy

Correlative light-electron microscopy (CLEM) unifies the versatility of light microscopy (LM) with the high resolution of electron microscopy (EM), allowing one to zoom into the complex organization of cells. Most CLEM techniques use ultrathin sections, and thus lack the 3D-EM structural information, and focusing on a very restricted field of view. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) microscopy over large field of view and high precision localization of the target area of interest within EM. The chip-based direct stochastic optical reconstruction microscopy (dSTORM), and 3D high precision correlation of biological processes by focused ion beam-scanning electron microscopy (FIB-SEM) is further demonstrated. The core layer of the photonic chips are used as a substrate to hold, to illuminate and the cladding layer is used to enable high-precision landmarking of the sample through specially designed grid-like numbering systems. The landmarks are fabricated on the cladding of the photonic chips as extruding pillars from the waveguide surface, thus remaining visible for FIB-SEM after resin embedding during sample processing. Using this approach we demonstrate its applicability for tracking the area of interest, imaging the 3D structural organization of nano-sized morphological features on liver sinusoidal endothelial cells such as fenestrations, and correlating specific endo-lysosomal compartments with its cargo protein upon endocytosis. We envisage that photonic chip equipped with landmarks can be used in the future to automatize the work-flow for both LM and EM for high-throughput CLEM, providing the resolution needed for insights into the complex intracellular communication and the relation between morphology and function in health and disease.

physics.optics↗