Cargando…

Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), m...

Descripción completa

Detalles Bibliográficos
Autores principales: Saitou, Takashi, Imamura, Takeshi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10298976/
https://www.ncbi.nlm.nih.gov/pubmed/37373345
http://dx.doi.org/10.3390/ijms241210186
_version_ 1785064249410715648
author Saitou, Takashi
Imamura, Takeshi
author_facet Saitou, Takashi
Imamura, Takeshi
author_sort Saitou, Takashi
collection PubMed
description Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution.
format Online
Article
Text
id pubmed-10298976
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-102989762023-06-28 Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution Saitou, Takashi Imamura, Takeshi Int J Mol Sci Article Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution. MDPI 2023-06-15 /pmc/articles/PMC10298976/ /pubmed/37373345 http://dx.doi.org/10.3390/ijms241210186 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Saitou, Takashi
Imamura, Takeshi
Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution
title Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution
title_full Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution
title_fullStr Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution
title_full_unstemmed Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution
title_short Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution
title_sort extended depth of focus two-photon light-sheet microscopy for in vivo fluorescence imaging of large multicellular organisms at cellular resolution
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10298976/
https://www.ncbi.nlm.nih.gov/pubmed/37373345
http://dx.doi.org/10.3390/ijms241210186
work_keys_str_mv AT saitoutakashi extendeddepthoffocustwophotonlightsheetmicroscopyforinvivofluorescenceimagingoflargemulticellularorganismsatcellularresolution
AT imamuratakeshi extendeddepthoffocustwophotonlightsheetmicroscopyforinvivofluorescenceimagingoflargemulticellularorganismsatcellularresolution