Close
Close

preLights is supported by…

Related posts

Related preLists

Extended depth of focus multiphoton microscopy via incoherent pulse splitting

Bingying Chen, Tonmoy Chakraborty, Stephan Daetwyler, James D. Manton, Kevin Dean, Reto Fiolka

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

Reaching deeper: multiphoton microscopy and incoherent pulse splitting

Selected by Mariana De Niz

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.

Figure 1. Extended depth of focus multiphoton microscopy via incoherent pulse splitting (Used with permission from ref. 1).

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

  1. Bingying Chen, Tonmoy Chakraborty, Stephan Daetwyler, James D. Manton, Kevin Dean, Reto Fiolka, Extended depth of focus multiphoton microscopy via incoherent pulse splitting, 2020, bioRxiv, doi: 10.1101/2020.03.27.012260

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 preprint (No Ratings Yet)

Author's response

Reto Fiolka, Bingying Chen, Tonmoy Chakraborty, Stephan Daetwyler, James Manton, and Kevin Dean shared

Open questions  

1.The main advantage of your work is rapid volumetric imaging. In your discussion, you mention as an example, that the increased depth of focus could be helpful in scenarios of significant sample motion. What is the maximum imaging speed you could achieve, and is this enough to visualize most phenomena occurring in vivo- for instance blood circulation?

We have not applied this technology to very rapid raster scanning microscopy, as our microscope does not possess resonant galvanometric scanners. However, in theory, the EDF is compatible with resonant scanning, in particular also with recently developed Lissajous scanning in two dimensions. Thereby, we anticipate frame rates as fast as 100-1000 Hz are feasible, provided the sample is bright enough.

 

2. You discuss also that your method is advantageous when multiple objects at different depths must be monitored simultaneously. What range of fluorophores/wavelengths can you visualize simultaneously?

Currently, we employ a Ti:Sapph laser in our microscope with a tuning range from 650-1050 nm, and we typically use 900 nm illumination to excite GFP. Unfortunately, tuning the laser is quite slow, so if we want to image a second channel, we typically use a fluorescent protein that can be excited at the same wavelength as GFP, but emits at a wavelength that can be separated with dichroics and emission filters.  Two great examples include CyOFP and LSS-mOrange.

If tuning the laser to a different excitation wavelength is not a problem, e.g. if you have two ultrafast lasers (each emitting at a different wavelength), or if you have an optical parametric oscillator, then in principle, you can use any fluorophore so long as it possesses a good two-photon absorption cross-section.  This is expensive, but feasible.

 

3. What were your findings in terms of phototoxicity and photobleaching compared with conventional two-photon imaging?

This is an interesting question that we have not yet studied. There are conflicting reports in the literature about the effects of phototoxicity and photobleaching, and their relation to peak power and the laser repetition-rate.  In general, it is our suspicion that decreasing the instantaneous laser power and integrating over a larger volume will decrease photobleaching and phototoxicity.  However, it is not clear if this effect would be measureable in our setup.

 

4. You mention in your discussion that existing two-photon raster scanning microscopes can be easily retrofitted without adding any other optical components than the mask. A big advantage of some commercially available microscopes is the possibility to easily switch modalities. Can the microscope be used in either mode, or how easy is it to switch?

To switch between the modes in an automated fashion, most likely a mechanical actuator could be used to swing the phase mask in and out of the optical train of the microscope.

 

5.Can you expand further why Bessel beams are robust to aberrations and optical occlusions, and how the EDF beam design can achieve this?

The assumption is that a Bessel beam is self-healing, as there is always new light entering the focal region from the periphery. Our EDF beam can be interpreted as a superposition of multiple Bessel beams, so the assumption is that each would inherit some of these self-healing properties. However, in what regimes this self-healing property really works is contested for Bessel beams themselves. For example, if you aberrate the plane wave that precedes the Bessel beam with strong aberrations, the Bessel beam itself will be severely aberrated. Also, it does not appear that a Bessel beam would be immune to strong light-scattering in tissues.  Thus, we believe that in the presence of strong light-scattering and aberrations, our focus will be as severely affected as normal laser foci.

 

6.How do the images acquired using your setup compare in terms of resolution to conventional confocal microscopy and conventional two-photon microscopy?

The most obvious difference is the increased depth of focus – which will cause more objects to appear sharply. However, there is a slight lateral deterioration of image resolution, theoretically 10%. So, a conventional two-photon volume will be slightly sharper laterally. Going to confocal microscopy, this will depend on the sample. A cell on a coverslip will likely be sharper imaged with a confocal. However, deeper inside a tissue, I expect that the EDF two-photon image will appear sharper. We expect that this cross-over point happens at 1-2 scattering mean free paths.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the biophysics category:

Small extracellular vesicles promote stiffness-mediated metastasis

Alexandra Sneider, Ying Liu, Bartholomew Starich, et al.



Selected by Jade Chan, yohalie kalukula

Frictiotaxis underlies adhesion-independent durotaxis

Adam Shellard, Peter A. E. Hampshire, Namid R. Stillman, et al.



Selected by Girish Kale

Phosphorylation controls spatial and temporal activities of motor-PRC1 complexes to complete mitosis

Agata Gluszek-Kustusz, Benjamin Craske, Thibault Legal, et al.



Selected by Divya Pathak, Barbora Knotkova

Also in the cell biology category:

TLR2 Regulates Hair Follicle Cycle and Regeneration via BMP Signaling

Luyang Xiong, Irina Zhevlakova, Xiaoxia Z. West, et al.



Selected by Marina Schernthanner

Mechano-dependent sorbitol accumulation supports biomolecular condensate

Stephanie Torrino, William M Oldham, Andrés R Tejedor, et al.



Selected by Teodora Piskova

CRISPR-dependent base editing screens identify separation of function mutants of RADX with altered RAD51 regulatory activity

Madison B. Adolph, Atharv S. Garje, Swati Balakrishnan, et al.



Selected by Jessica Chevallier, Pierre Caron

preLists in the biophysics category:

66th 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
Close