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Label retention expansion microscopy (LR-ExM) with Aydin denoising

Speakers: Xiang Zhao and Ahmet Can Solak Moderator: Xiaoyu Shi

Online live: Nov 08, 2022 12:00 PM EST Views: 2735

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Abstract

Expansion microscopy (ExM) developed in the recent decade has enabled conventional confocal microscopy to be used for super-resolution imaging by physically expanding the sample within swellable hydrogel. Since its invention, ExM has been tested with many different types of biological samples. The main challenge addressed frequently is the fluorescence signal loss during polymerization and digestion of ExM protocols. The recently developed label-retention ExM has solved the problem with trifunctional anchors. LR-ExM has enabled high-efficiency labeling and prevented the signaling loss, but it has also increased the signaling noises meanwhile. Here we have established the protocol for applying LR-ExM with zebrafish cryosections at first. Together with Aydin denoising, we have optimized the imaging data set and obtained high quality super-resolution immunofluorescent images. Leverage on both LR-ExM with Aydin denoising tools, we have identified that cytosolic Par-3 is associated with Dynein complex and Dld on Rab11 endosomes in the mitotic radial glia cells in zebrafish forebrain.

Speakers

Xiang Zhao

Xiang Zhao, Ph.D.

Research Scientist, Chan Zuckerberg Biohub / University of California San Francisco

Xiang Zhao is a neuroscientist enthusiastic about the gene function and regulation mechanism in the development of the central nervous system. Xian...

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Ahmet Can Solak

Ahmet Can Solak, BSc.

Software Engineer, Chan Zuckerberg Biohub

Ahmet Can Solak, got his BSc. on Electrical and Electronics Engineering in 2018 from Koc University in Istanbul, Turkey. He joined the Loic Royer’s...

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Moderator

Xiaoyu Shi

Xiaoyu Shi, Ph.D.

Assistant Professor, University of California

Xiaoyu Shi is an Assistant Professor in the Department of Developmental and Cell Biology and the Department of Chemistry at the University of Calif...

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Keywords

Expansion microscopy, Aydin imaging denoising, Par-3, Endosome, Asymmetric cell division

References

1.

Zhao, X., Garcia, J.Q., Royer, L.A., and Guo, S. (2022) Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP. Bio-protocol 12(5): e4336. PMCID: PMC8918214.

2.
Zhao X, Garcia JQ, Tong K, Chen X, Yang B, Li Q, Dai Z, Shi X, Seiple IB, Huang B, Guo S. Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division. Sci Adv. 2021 Jun 11;7(24):eabg1244. doi: 10.1126/sciadv.abg1244. PMID: 34117063; PMCID: PMC8195473.
3.
Aydin: image denoising but chill. DOI 10.5281/zenodo.5654826
4.
Ahmet Can Solat: DaXi—high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy. Yang et al., Nature Methods, April 2022
5.
Democratising deep learning for microscopy with ZeroCostDL4Mic}{ Chamier and Laine et al., Nature Communications (April 2021)
6.
Image deconvolution via noise-tolerant self-supervised inversion. Hirofumi Kobayashi, Ahmet Can Solak, Joshua Batson, Loic A. Royer, ArXiv (June 2020)
7.
Shi X, Li Q, Dai Z, Tran AA, Feng S, Ramirez AD, Lin Z, Wang X, Chow TT, Chen J, Kumar D, McColloch AR, Reiter JF, Huang EJ, Seiple IB, Huang B. Label-retention expansion microscopy. J Cell Biol. 2021 Sep 6;220(9):e202105067. doi: 10.1083/jcb.202105067. Epub 2021 Jul 6. PMID: 34228783; PMCID: PMC8266563.

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22 Q&A

Dear Sir, My research field is studies of chromosomes and spore morphology in Pteridophytes. Will it help for my research work?

This microscope is use Only for genetically level research or others?

edit 2 Answers 50 Views Oct 29, 2022
XZ Xiang Zhao

Label-retention Expansion microscopy are mainly used for imaging cell organelles at super-resolution by physically expanding of the sample in each direction evenly with the embedding of hydrogel. If the objects are visible by light microscopes, you may not need to do expansion microscopy then.

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helpful 4 helpful
PV Periyasamy Vijayakanth

Thank you Sir.

helpful 0 helpful
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novelty chemistry availability?

how widely available are some of these specialty chemicals for the crosslinking/labeling?

edit 1 Answer 21 Views Oct 28, 2022
XZ Xiang Zhao

All reagents used for ExM are available from commercial vendors. Please find details from the reference: Shi X, Li Q, Dai Z, Tran AA, Feng S, Ramirez AD, Lin Z, Wang X, Chow TT, Chen J, Kumar D, McColloch AR, Reiter JF, Huang EJ, Seiple IB, Huang B. Label-retention expansion microscopy. J Cell Biol. 2021 Sep 6;220(9):e202105067. doi: 10.1083/jcb.202105067. Epub 2021 Jul 6. PMID: 34228783; PMCID: PMC8266563.

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What is standard sample preparation protocol for super resolution microscopy?

?What is standard sample preparation protocol for super resolution microscopy ?

edit 1 Answer 20 Views Oct 28, 2022
XZ Xiang Zhao

Most are very similar. You can check the protocol we have used in follow publications:


Zhao, X., Garcia, J.Q., Royer, L.A., and Guo, S. (2022) Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP. Bio-protocol 12(5): e4336. PMCID: PMC8918214.


Shi X, Li Q, Dai Z, Tran AA, Feng S, Ramirez AD, Lin Z, Wang X, Chow TT, Chen J, Kumar D, McColloch AR, Reiter JF, Huang EJ, Seiple IB, Huang B. Label-retention expansion microscopy. J Cell Biol. 2021 Sep 6;220(9):e202105067. doi: 10.1083/jcb.202105067. Epub 2021 Jul 6. PMID: 34228783; PMCID: PMC8266563.

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helpful 3 helpful
Join Discussion

Is the webinar cancelled I cannot access the video link?

Is the webinar cancelled I cannot access the video link?

Xiaoqing Qu
follow or recommend this question
edit 0 Answer 9 Views Nov 9, 2022

How efficient the technique is to work with primary and fluorophore tagged secondary antibodies?

I am new to expansion microscopy and have never tried in my hand.

Let's say in case when someone don't have access to tagged protein. And want to visualize endogenous protein using the primary antibodies and further to visualize them use any fluorophore tagged secondary antibody. In that case how efficient the system is. Especially when it comes to signal/noise ration.

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edit 0 Answer 9 Views Oct 28, 2022

1. How this technique is superior from darkfield microscope for fluorescent labelled complex in tissue sections?

I want to know about this technique that it is superior than darkfield microscopy technique commonly used for the visualization of fluorescent labelled complex in different tissue section

edit 0 Answer 7 Views Oct 31, 2022

can we get (where can we get) the trifunctional probes for LR-ExM?

The key component for LR-ExM is the trifuncitonal probe. Is it now commercially available? or how can someone (i.e. us) get some of the probe?

edit 0 Answer 6 Views Nov 2, 2022

What are some of you preferred markers to estimate the degree of expansion?

what are the preferred methods to determine the degree of expansion? Exogenous Fiducial markers, naturally occurring structures inner and outer membranes of the mitochondria etc. Should this be done every time?

edit 0 Answer 6 Views Oct 28, 2022

how it works,the future of this technology industry, and its promises?

This is my first time hearing about this technology so I would like to know how it works,the future of this technology industry, and its promises it holds for the healthcare domain.

edit 0 Answer 5 Views Oct 28, 2022

Is ExM suitable to search for cell-cell contacts in tissues?

I am investigating extra-embryonic membranes (serosa and amnion) in red flour beetle, Tribolium castaneum. Both membranes are single layer epithelial sheets stretched on the surface of an ellipsoid egg (600 um in length, 250 um in diameter). Serosa is the outer membrane, amnion is the inner layer, their basal sides facing each other. I am looking for cell-cell contacts between these membranes. It is unclear if there is any. I would like to use ExM instead of TEM.

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edit 0 Answer 4 Views Nov 8, 2022

I want to know is this technology is useful for cross section studies for cereal seed?

We want to visualize the developing pigmentation pattern in wheat seed. So please suggest the fluorescent dye that can be useful for the same.

edit 0 Answer 4 Views Oct 28, 2022

What should you pay attention to when using for the first time?

Does the sample to be observed require any special treatment? What special attention should be paid to the process?

edit 0 Answer 3 Views Nov 7, 2022

Is there an article describing Aydin denoising?

I am interested in learning more about the origin of this denoising method.

edit 0 Answer 3 Views Nov 4, 2022

How could we use LR- ExM with cell markers to identify cell types?

I am dealing with Neurons and Glial cell culture. I am using markers to visualize the cells. like GFAP is used to identify astroglia. so, I wanted to know, how could I use this LR- Exm with my study?

edit 0 Answer 3 Views Nov 2, 2022

Did LR-ExM will be helpful in analyzing aptamer tagged gene based drug effect in biological systems and it's proteomics changes?

1) In a study for Gene based drug action in cells or rodents, can we check changes occurring at the molecular level through LR-EXm?

2) whether it is compulsory to have drug fluorescent leveled or not for detection?

3) Can we do kinetics study using LR-EXm?

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edit 0 Answer 3 Views Oct 28, 2022

Applications of the microscopy, and why it is superior than other microscopy?

I am a beginner and want to know abot this technology.

edit 0 Answer 3 Views Oct 28, 2022

Why lr- exm microposcopy is more effective than other kinds of microscopy?

what are the main advantages and uses of lr-exm microscopy?

edit 0 Answer 3 Views Oct 28, 2022

How much is this technology beneficial for research?

I would like to learn in depth about this technology

edit 0 Answer 3 Views Oct 28, 2022

what are the clinical application of Label Retention Expansion Microscopy?

what are the clinical application of Label Retention Expansion Microscopy


edit 0 Answer 2 Views Oct 27, 2022

What are the usages of this technolgy in pharmacolgy?

For example in cancer, neuroscience, herbalmedicine, and etc... .

edit 0 Answer 4 Views Oct 28, 2022

What types of tinction can be used in this kind of Microscopy?

This can be a sofisticated method. Other products can be used in the technique that are used in other microscopy techniques?

edit 0 Answer 3 Views Oct 27, 2022

New advanced and recent molecular techniques techniques?

All advanced way of microbes isolation

edit 0 Answer 6 Views Oct 28, 2022