Light-sheet microscopes have become the tool of choice for volumetric imaging of large samples. Based on a wide-field acquisition scheme, they are capable of optical sectioning at diffraction-limited resolution and minimal overall photodamage. Unfortunately, traditional architectures are limited in speed because 3D images are collected by either sample translation or synchronized movement of both light-sheet and detection objective lens. A promising solution avoiding slow mechanical movements is to extend the depth-of-field of the microscope and moving only the light-sheet. However, this normally comes at the cost of losing light and contrast, compromising the signal-to-noise ratio of the images. Here, we propose an innovative technique devoted to restoring the quality of the images, while preserving the speed of extended depth-of-field microscopes. It is based on generating a stack of parallel light-sheets using a pair of orthogonal acousto-optic deflectors, enabling the simultaneous illumination of different sample planes. Given the extended depth-of-field, all such planes appear in focus and can be acquired in a superimposed single frame. By applying a single-step inversion algorithm, we can decode a stack of frames into a volumetric image whose signal-to-noise ratio and contrast are greatly enhanced. We provide a detailed theoretical framework of the method and demonstrate its feasibility with volumetric images of kidney cell spheroids.

Multiplane Encoded Light-Sheet Microscopy for Enhanced 3D Imaging

Zunino A.;Trianni A.;Diaspro A.;
2021-01-01

Abstract

Light-sheet microscopes have become the tool of choice for volumetric imaging of large samples. Based on a wide-field acquisition scheme, they are capable of optical sectioning at diffraction-limited resolution and minimal overall photodamage. Unfortunately, traditional architectures are limited in speed because 3D images are collected by either sample translation or synchronized movement of both light-sheet and detection objective lens. A promising solution avoiding slow mechanical movements is to extend the depth-of-field of the microscope and moving only the light-sheet. However, this normally comes at the cost of losing light and contrast, compromising the signal-to-noise ratio of the images. Here, we propose an innovative technique devoted to restoring the quality of the images, while preserving the speed of extended depth-of-field microscopes. It is based on generating a stack of parallel light-sheets using a pair of orthogonal acousto-optic deflectors, enabling the simultaneous illumination of different sample planes. Given the extended depth-of-field, all such planes appear in focus and can be acquired in a superimposed single frame. By applying a single-step inversion algorithm, we can decode a stack of frames into a volumetric image whose signal-to-noise ratio and contrast are greatly enhanced. We provide a detailed theoretical framework of the method and demonstrate its feasibility with volumetric images of kidney cell spheroids.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1075148
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 4
  • ???jsp.display-item.citation.isi??? 4
social impact