Computational multifocus fluorescence microscopy for three-dimensional visualization of multicellular tumor spheroids

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract.

Significance: Three-dimensional (3D) visualization of multicellular tumor spheroids (MCTS) in fluorescence microscopy can rapidly provide qualitative morphological information about the architecture of these cellular aggregates, which can recapitulate key aspects of their in vivo counterpart.

Aim: The present work is aimed at overcoming the shallow depth-of-field (DoF) limitation in fluorescence microscopy while achieving 3D visualization of thick biological samples under study.

Approach: A custom-built fluorescence microscope with an electrically focus-tunable lens was developed to optically sweep in-depth the structure of MCTS. Acquired multifocus stacks were combined by means of postprocessing algorithms performed in the Fourier domain.

Results: Images with relevant characteristics as extended DoF, stereoscopic pairs as well as reconstructed viewpoints of MCTS were obtained without segmentation of the focused regions or estimation of the depth map. The reconstructed images allowed us to observe the 3D morphology of cell aggregates.

Conclusions: Computational multifocus fluorescence microscopy can provide 3D visualization in MCTS. This tool is a promising development in assessing the morphological structure of different cellular aggregates while preserving a robust yet simple optical setup.

Related collections

Most cited references 38

Record: found
Abstract: found
Article: not found

Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.

Bi-Chang Chen, Wesley R Legant, Kai Wang … (2014)

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems. Copyright © 2014, American Association for the Advancement of Science.

0 comments Cited 382 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Wide-field, high-resolution Fourier ptychographic microscopy

Guoan Zheng, Roarke Horstmeyer, Changhuei Yang (2013)

In this article, we report an imaging method, termed Fourier ptychographic microscopy (FPM), which iteratively stitches together a number of variably illuminated, low-resolution intensity images in Fourier space to produce a wide-field, high-resolution complex sample image. By adopting a wavefront correction strategy, the FPM method can also correct for aberrations and digitally extend a microscope’s depth-of-focus beyond the physical limitations of its optics. As a demonstration, we built a microscope prototype with a resolution of 0.78 μm, a field-of-view of ~120 mm2, and a resolution-invariant depth-of-focus of 0.3 mm (characterized at 632 nm). Gigapixel colour images of histology slides verify FPM’s successful operation. The reported imaging procedure transforms the general challenge of high-throughput, high-resolution microscopy from one that is coupled to the physical limitations of the system’s optics to one that is solvable through computation.

0 comments Cited 302 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Fluorescence microscopy.

Jeff W. Lichtman, José-Angel Conchello (2005)

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.

0 comments Cited 200 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Julia R. Alonso

Alejandro Silva

Ariel Fernández

Miguel Arocena:

ORCID: https://orcid.org/0000-0002-7682-4028

Journal

Journal ID (nlm-ta): J Biomed Opt

Journal ID (iso-abbrev): J Biomed Opt

Journal ID (coden): JBOPFO

Journal ID (publisher-id): JBO

Title: Journal of Biomedical Optics

Publisher: Society of Photo-Optical Instrumentation Engineers

ISSN (Print): 1083-3668

ISSN (Electronic): 1560-2281

Publication date (Electronic): 2 June 2022

Publication date (Print): June 2022

Publication date PMC-release: 2 June 2022

Volume: 27

Issue: 6

Electronic Location Identifier: 066501

Affiliations

[a ]Universidad de la República , Instituto de Física, Facultad de Ingeniería, Montevideo, Uruguay

[b ]Instituto de Investigaciones Biológicas Clemente Estable , Departamento de Genómica, Montevideo, Uruguay

[c ]Universidad de la República , Cátedra de Bioquímica y Biofísica, Facultad de Odontología, Montevideo, Uruguay

Author notes

[* ]Address all correspondence to Julia R. Alonso, julialon@ 123456fing.edu.uy

Author information

Miguel Arocena https://orcid.org/0000-0002-7682-4028

Article

Publisher-manuscript ID: JBO-210320SSR Publisher ID: 210320SSR

DOI: 10.1117/1.JBO.27.6.066501

PMC ID: 9162503

PubMed ID: 35655357

SO-VID: f5426886-d7f2-4437-8c1e-1d14e03ca354

License:

Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

History

Date received : 18 October 2021

Date accepted : 23 May 2022

Page count

Figures: 5, Tables: 1, References: 38, Pages: 11

Funding

Funded by: Comision Sectorial de Investigacion Cientfica - CSIC - Uruguay

Award ID: ID - 237

Custom metadata

running-head Alonso et al.: Computational multifocus fluorescence microscopy for three-dimensional visualization…

ScienceOpen disciplines: Biomedical engineering

Keywords: fluorescence microscopy,three-dimensional visualization,stereoscopic pairs,computational optical imaging

Data availability:

ScienceOpen disciplines: Biomedical engineering

Keywords: fluorescence microscopy, three-dimensional visualization, stereoscopic pairs, computational optical imaging

Comments

Comment on this article

scite_

Smart Citations

Citing PublicationsSupportingMentioningContrasting

View Citations

See how this article has been cited at scite.ai

scite shows how a scientific paper has been cited by providing the context of the citation, a classification describing whether it supports, mentions, or contrasts the cited claim, and a label indicating in which section the citation was made.