Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy

Year: 2023

Authors: Gardini L., Vignolini T., Curcio V., Pavone FS., Capitanio M.

Autors Affiliation: CNR, Natl Inst Opt, Via N Carrara 1, I-50019 Sesto Fiorentino, Italy; LENS, European Lab Nonlinear Spect, Via N Carrara 1, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Phys & Astron, Via G Sansone 1, I-50019 Sesto Fiorentino, Italy.

Abstract: In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super -resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super -resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far-and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 & mu;m). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 & mu;m while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds. & COPY; 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement Journal/Review: OPTICS EXPRESS

Volume: 31 (16)      Pages from: 492152-1  to: 492152-18

More Information: Ministry of University and Research (MUR) (Ministero dell’Universita & nbsp;e della Ricerca) , National Recovery and Reslience Plan (NRRP) , (project MNESYS (PE0000006) ; European Union’s Horizon 2020 Framework Programme grant agreement no (871124, Laserlab-Europe) .
KeyWords: Single; Dna; Molecules; Tracking
DOI: 10.1364/OE.492152

ImpactFactor: 3.200
Citations: 2
data from “WEB OF SCIENCE” (of Thomson Reuters) are update at: 2024-11-24
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