Nanoparticle Suspensions Enclosed in Methylcellulose: a New Approach for Quantifying Nanoparticles in Transmission Electron Microscopy

Overview

Journal Sci Rep

Specialty Science

Date 2016 May 5

PMID 27141843

Citations 10

Authors

Christian Hacker

Jalal Asadi

Christos Pliotas

Sophie Ferguson

Lee Sherry

Phedra Marius

Javier Tello

David Jackson

James Naismith

John Milton Lucocq

Affiliations

Soon will be listed here.

Abstract

Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM.

Citing Articles

Functional redundancy revealed by the deletion of the mimivirus GMC-oxidoreductase genes.

Alempic J, Bisio H, Villalta A, Santini S, Lartigue A, Schmitt A Microlife. 2024; 5:uqae006.

PMID: 38659623 PMC: 11042495. DOI: 10.1093/femsml/uqae006.

Characterization and Vaccine Potential of Outer Membrane Vesicles from subsp. .

Teixeira A, Loureiro I, Lisboa J, Oliveira P, Azevedo J, Dos Santos N Int J Mol Sci. 2023; 24(6).

PMID: 36982212 PMC: 10049053. DOI: 10.3390/ijms24065138.

Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.

Hacker C, Schrader T, Schrader M Methods Mol Biol. 2023; 2643:105-122.

PMID: 36952181 DOI: 10.1007/978-1-0716-3048-8_8.

Bone Marrow Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles Promote Corneal Wound Repair by Regulating Inflammation and Angiogenesis.

Saccu G, Menchise V, Gai C, Bertolin M, Ferrari S, Giordano C Cells. 2022; 11(23).

PMID: 36497151 PMC: 9736484. DOI: 10.3390/cells11233892.

Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in live single cells by site-specific direct stochastic optical reconstruction microscopy.

Chakkarapani S, Shin T, Lee S, Park K, Lee G, Kang S J Nanobiotechnology. 2021; 19(1):398.

PMID: 34844629 PMC: 8628397. DOI: 10.1186/s12951-021-01147-1.

References

van der Pol E, Coumans F, Varga Z, Krumrey M, Nieuwland R . Innovation in detection of microparticles and exosomes. J Thromb Haemost. 2013; 11 Suppl 1:36-45. DOI: 10.1111/jth.12254. View

Brydson R, Brown A, Hodges C, Abellan P, Hondow N . Microscopy of nanoparticulate dispersions. J Microsc. 2015; 260(3):238-47. DOI: 10.1111/jmi.12290. View

Jain S, Doshi A, Iyer A, Amiji M . Multifunctional nanoparticles for targeting cancer and inflammatory diseases. J Drug Target. 2013; 21(10):888-903. DOI: 10.3109/1061186X.2013.832769. View

Pekkurnaz G, Fera A, Zimmerberg-Helms J, DeGiorgis J, Bezrukov L, Blank P . Isolation and ultrastructural characterization of squid synaptic vesicles. Biol Bull. 2011; 220(2):89-96. PMC: 3548571. DOI: 10.1086/BBLv220n2p89. View

Tokuyasu K . A study of positive staining of ultrathin frozen sections. J Ultrastruct Res. 1978; 63(3):287-307. DOI: 10.1016/s0022-5320(78)80053-7. View

Inagaki S, Ghirlando R, Grisshammer R . Biophysical characterization of membrane proteins in nanodiscs. Methods. 2012; 59(3):287-300. PMC: 3608844. DOI: 10.1016/j.ymeth.2012.11.006. View

Pliotas C, Dahl A, Rasmussen T, Mahendran K, Smith T, Marius P . The role of lipids in mechanosensation. Nat Struct Mol Biol. 2015; 22(12):991-8. PMC: 4675090. DOI: 10.1038/nsmb.3120. View

Wang R, Pokhariya H, McKenna S, Lucocq J . Recognition of immunogold markers in electron micrographs. J Struct Biol. 2011; 176(2):151-8. DOI: 10.1016/j.jsb.2011.07.005. View

Weon B, Je J . Capillary force repels coffee-ring effect. Phys Rev E Stat Nonlin Soft Matter Phys. 2010; 82(1 Pt 2):015305. DOI: 10.1103/PhysRevE.82.015305. View

10.

Dragovic R, Gardiner C, Brooks A, Tannetta D, Ferguson D, Hole P . Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine. 2011; 7(6):780-8. PMC: 3280380. DOI: 10.1016/j.nano.2011.04.003. View

11.

Hinuma Y, Konn M, Yamaguchi J, GRACE Jr J . Replication of herpes-type virus in a Burkitt lymphoma cell line. J Virol. 1967; 1(6):1203-6. PMC: 375410. DOI: 10.1128/JVI.1.6.1203-1206.1967. View

12.

Tyrrell D, Valentine R . The assay of influenza virus particles by haemagglutination and electron microscopy. J Gen Microbiol. 1957; 16(3):668-75. DOI: 10.1099/00221287-16-3-668. View

13.

Lucocq J . Can data provenance go the full monty?. Trends Cell Biol. 2012; 22(5):229-30. DOI: 10.1016/j.tcb.2012.03.001. View

14.

Momen-Heravi F, Balaj L, Alian S, Mantel P, Halleck A, Trachtenberg A . Current methods for the isolation of extracellular vesicles. Biol Chem. 2013; 394(10):1253-62. PMC: 7075462. DOI: 10.1515/hsz-2013-0141. View

15.

Cheng Y, Morshed R, Auffinger B, Tobias A, Lesniak M . Multifunctional nanoparticles for brain tumor imaging and therapy. Adv Drug Deliv Rev. 2013; 66:42-57. PMC: 3948347. DOI: 10.1016/j.addr.2013.09.006. View

16.

Kourembanas S . Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol. 2014; 77:13-27. DOI: 10.1146/annurev-physiol-021014-071641. View

17.

Anderson N, Doane F . Agar diffusion method for negative staining of microbial suspensions in salt solutions. Appl Microbiol. 1972; 24(3):495-6. PMC: 376545. DOI: 10.1128/am.24.3.495-496.1972. View

18.

De Carlo S, Harris J . Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron. 2010; 42(2):117-31. PMC: 2978762. DOI: 10.1016/j.micron.2010.06.003. View

19.

Tokuyasu K . Immunochemistry on ultrathin frozen sections. Histochem J. 1980; 12(4):381-403. DOI: 10.1007/BF01011956. View

20.

Newcomb C, Moyer T, Lee S, Stupp S . Advances in cryogenic transmission electron microscopy for the characterization of dynamic self-assembling nanostructures. Curr Opin Colloid Interface Sci. 2012; 17(6):350-359. PMC: 3510006. DOI: 10.1016/j.cocis.2012.09.004. View