Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System

Mondal, Prasenjit; Chowdhury, Rajdeep; Nandi, Shyamapada; Amin, Md. Asif; Bhattacharyya, Kankan; Ghosh, Surajit

doi:10.1002/asia.201900588

Cited by 5 publications

(4 citation statements)

References 56 publications

(112 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Apart from applications in imaging protein distributions, it can also be important to visualize cellular structures by means of lipid staining. Biotin-DHPE can be used to label lipid-containing structures 35 – 37 , so we investigated if it can be applied to TT-ExM to stain the lipid structures of cells. We dissolved biotin-DHPE in chloroform and then diluted the mix with 70% or 50% ethanol for cell staining, with incubation times of 150 min or overnight.…”

Section: Resultsmentioning

confidence: 99%

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Wang,

Tian,

Liou

et al. 2023

Sci Rep

View full text Add to dashboard Cite

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.

show abstract

Section: Resultsmentioning

confidence: 99%

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Wang,

Tian,

Liou

et al. 2023

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Apart from applications in imaging protein distributions, it can also be important to visualize cellular structures by means of lipid staining. Biotin-DHPE can be used to label lipid-containing structures [16][17][18], so we investigated if it can be applied to TT-ExM to stain the lipid structures of cells. We dissolved biotin-DHPE in chloroform and then diluted the mix with 70% or 50% ethanol for cell staining, with incubation times of 150 min or overnight.…”

Section: Application Of Tt-exm For Lipid Stainingmentioning

confidence: 99%

Expansion microscopy with trypsin digestion and tyramide signal amplification (TT-ExM) for protein and lipid staining

Wang

Tian

Liou³

et al. 2023

Preprint

View full text Add to dashboard Cite

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers two major drawbacks, namely proteolysis and swelling effects that, respectively, induce protein loss and dilute fluorescence signals. Here, we report two improvements to expansion microscopy that overcome these two challenges, i.e., deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. Notably, we demonstrate better protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures following 2-h trypsin treatment. Subsequent lipid staining revealed the complex 3D membrane structures in entire cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. Thus, TT-ExM significantly enhances fluorescent signals and generates high-quality and ultrafine-resolution images under confocal microscopy.

show abstract

“…Apart from applications in imaging protein distributions, it can also be important to visualize cellular structures by means of lipid staining. Biotin-DHPE can be used to label lipid-containing structures [35][36][37] , so we investigated if it can be applied to TT-ExM to stain the lipid structures of cells. We dissolved biotin-DHPE in chloroform and then diluted the mix with 70% or 50% ethanol for cell staining, with incubation times of 150 min or overnight.…”

Section: Application Of Tt-exm For Lipid Stainingmentioning

confidence: 99%

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Wang,

Tian,

Liou

et al. 2023

Preprint

View full text Add to dashboard Cite

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase and lipid-rich chromosome matrices in the mitotic cells. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images.

show abstract

Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System

Cited by 5 publications

References 56 publications

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Expansion microscopy with trypsin digestion and tyramide signal amplification (TT-ExM) for protein and lipid staining

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Contact Info

Product

Resources

About