Extended depth of focus multiphoton microscopy via incoherent pulse splitting
Preprint posted on 29 March 2020 https://www.biorxiv.org/content/10.1101/2020.03.27.012260v1
Article now published in Biomedical Optics Express at http://dx.doi.org/10.1364/BOE.393931
Categories: biophysics, cell biology
Background
Imaging large volumes is necessary in order to understand biological phenomena that manifest themselves at the tissue- or organ-scale. However, imaging large volumes with raster scanning microscopes is very time consuming, particularly if using high NA objectives that provide a high lateral resolution and a small depth of focus. To capture volumetric imaging data, many planes have to be imaged by z-stepping each focal plane, which limits the achievable imaging speed considerably. Instead, if axial image formation can be sacrificed, extending the depth of focus (EDF) would allow the volumetric information to be projected into a single 2D image. This is especially attractive for rapid imaging of sparsely populated structures.
Key findings and developments
In their work, the authors present a phase mask that can be easily added to any multi-photon raster scanning microscope to extend the depth of focus five-fold with a small penalty in lateral resolution. Previous work designed for fluorescence widefield microscopy used an incoherent superposition as the means of creating an EDF beam: namely, the pupil of an objective was segmented into multiple annuli, which became incoherent to each other after passing through the mask. In their work, the authors applied this concept to beam shaping of ultrafast laser sources, achieving two-photon EDF microscopy by using a stepped annular phase mask. The phase mask, which is acting in a Fourier plane of the imaging system, consists of multiple concentric glass disks. An ultrafast laser pulse is split by the phase mask into different annular beamlets, each of which is time-delayed, forming a focus in the front focal plane of the objective at slightly different arrival times. The resulting EDF focus is the incoherent superposition of all individual foci. Numerical simulations to compute the electromagnetic field in the front plane of an objective were performed and predicted an extension of the depth of focus that scales with the number of annular zones. Finally, the authors demonstrated the potential of the EDF mask by imaging GFP-labelled neurons in fixed mouse brains. Altogether, the numerical and experimental results demonstrated that a five-fold extension in focal depth was feasible, with only a moderate lateral resolution loss.

What I like about this preprint
This exciting development could enable simultaneous high-speed imaging of biological phenomena at multiple depths. Examples include intravital microscopy or in situ imaging in tissues or whole cell contexts. Importantly, this advance can be incorporated into existing equipment with minimal modifications.
Reference
Acknowledgement
Many thanks to all authors for their engagement in discussions and questions.
Posted on: 16 April 2020
doi: https://doi.org/10.1242/prelights.18768
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the biophysics category:
Small extracellular vesicles promote stiffness-mediated metastasis
Frictiotaxis underlies adhesion-independent durotaxis
Phosphorylation controls spatial and temporal activities of motor-PRC1 complexes to complete mitosis
Also in the cell biology category:
TLR2 Regulates Hair Follicle Cycle and Regeneration via BMP Signaling
Mechano-dependent sorbitol accumulation supports biomolecular condensate
CRISPR-dependent base editing screens identify separation of function mutants of RADX with altered RAD51 regulatory activity
preListsbiophysics category:
in the66th Biophysical Society Annual Meeting, 2022
Preprints presented at the 66th BPS Annual Meeting, Feb 19 - 23, 2022 (The below list is not exhaustive and the preprints are listed in no particular order.)
List by | Soni Mohapatra |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
Biophysical Society Meeting 2020
Some preprints presented at the Biophysical Society Meeting 2020 in San Diego, USA.
List by | Tessa Sinnige |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Biomolecular NMR
Preprints related to the application and development of biomolecular NMR spectroscopy
List by | Reid Alderson |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
List by | Joseph Jose Thottacherry |
Also in the cell biology category:
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
List by | Dey Lab, Amanda Haage |