Visualizing Nanomedicines: Understanding Your Nanoparticle Formulations

NIS Mountain Hero
NIS Favicon
NanoImaging Services
May 30, 2023

x min read

Microscopic image of nanoparticle characterization with cryo-TEM from NanoImaging services

Many different techniques exist for studying and characterizing nanoparticles, including dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography.

In recent years, cryogenic transmission electron microscopy (cryo-TEM) has emerged as the “golden standard technology to observe particle size, morphology and potentially drug loading”1. It can visualize and measure multiple aspects of many kinds of samples, with only a small amount of sample needed. However, cryo-TEM requires expertise, and sample preparation can be challenging. Knowing the advantages and limitations to each technique can help you select the best analytical method for your sample or study.

Using Dynamic Light Scattering for Nanoparticle Characterization

Dynamic light scattering (DLS) is a widely used technique for nanoparticle characterization. DLS is a non-invasive, non-destructive technique that measures the hydrodynamic radius of particles in solution by analyzing the fluctuations in the scattered light caused by Brownian motion1. The technique is fast and easy to use, and is particularly useful for measuring the size distribution of nanoparticles in solution and can provide information on the stability and aggregation state of the particles. DLS has limitations in terms of the size range of particles it can measure, typically up to a few hundred nanometers1. Additionally, DLS assumes that the particles are spherical and homogenous, which may not be the case for some complex samples, and measurements can be affected by factors including temperature, concentration, and pH viscosity of the buffer2

A paper published in Science Direct in 2011 notes that “standard intensity-based DLS measurements have proved to be inadequate in determining the true number-weighted size distribution of heterogeneous liposome populations”2. Further, the paper states that “cryo-EM has become a leading technology for imaging nanoscale biological samples such as lipid nanoparticles” due to cryo-EM’s ability to visualize size, shape, morphology, lamellarity, and more in one study2.

Examples of particle sizing traces and particle diameter curves for HPV VLPs Nanoparticle characterization Nano Imaging Services
Example of particle size determination analysis tracing (left) and particle diameter curves (right) of HPV virus-like particles (VLPs).

Analytical Ultracentrifugation Sedimentation Velocity for Analyzing Sedimentation Behavior of Nanoparticles

Analytical ultracentrifugation sedimentation velocity (AUC-SV) is a technique used analyze the sedimentation behavior of nanoparticles in solution, primarily used to measure the ratio of full to empty capsids in virus-like particles (VLPs), especially in adeno-associated viruses (AAVs). This technique involves spinning a sample of nanoparticles at high speeds in a centrifuge and analyzing the resulting sedimentation profiles.  Nanoparticles can be characterized according to their size distribution, shape, density, and molecular weight using AUC. The method is particularly useful for studying the hydrodynamic behavior of nanoparticles and their interactions in solution. AUC, however, requires specialized equipment and expertise and can be time-consuming. AUC can require up to 400μl of sample, while cryo-TEM uses as little as 50μl of sample5. Cryo-TEM can also be used to determine particle size distribution, particle titer distribution, and more, as well as full/empty analysis, enabling you to gain more insight into your samples. To learn more about using cryo-TEM for AAV, read the whitepaper here.

In this paper in Molecular Therapy, Methods & Clinical Development, the authors compare AUC and cryo-TEM when assessing the capsid content of adeno-associated viruses (AAV)3. AUC was able to quantify expected capsid with high accuracy relative to the theoretical spiked levels and was able to resolve intermediate density. However AUC’s limitations include relatively low throughput data acquisitions, and compliance concerns related to instrument-specific acquisition software that makes AUC a technique that is not cGMP compliant3. NanoImaging Services plans offers cGMP compliant cryo-TEM studies, learn more now

Example image of cryo-TEM imaging showing full and empty capsids in adeno-associated virus (AAV)

Analyzing Nanoparticle Size Distribution with Size Exclusion Chromatography

microscopic black and white image of mRNA encapsulated bleb formations on lipid nanoparticle characterization with cryo-TEM from NanoImaging Services
Cryo-TEM image showing bleb compositions on lipid nanoaprticles (LNPs)

SEC is a technique used for the separation and purification of macromolecules based on their size. The technique involves passing a sample of nanoparticles through a column packed with porous beads. The larger nanoparticles are excluded from the pores and elute first, while smaller nanoparticles penetrate the pores and elute later. SEC can provide information on the size distribution, capsid content, molecular weight, and purity of nanoparticles. It is particularly useful for separating nanoparticles from impurities and can provide insights into their interactions in solution. In this study size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and cryo-TEM are compared for determining adeno-associated virus (AAV) capsid composition; SEC-MALS is unable to resolve intermediate capsids, whereas cryo-EM is able to do so3. Further, when analyzing lipid nanoparticles (LNPs), cryo-TEM is able to visualize blebs, whereas SEC does not give information about bleb composition, which is a key differentiator of LNPs from liposomes6.

Why is Cryo-TEM the best choice for Nanoparticle Characterization?

Cryo-TEM is a high-resolution imaging technique that allows for the direct observation of nanoparticles in their native state, and can visualize particles from 5 nm to 1 micron. Samples are flash-frozen at liquid nitrogen temperatures and imaged under a high-resolution transmission electron microscope. Cryo-TEM can provide detailed information on the size, morphology & shape, lamellarity, and internal structure of nanoparticles at the single-particle level. It is particularly useful for characterizing complex and heterogeneous samples, such as those with multiple components or surface modifications. 

Cryo-TEM requires specialized equipment and expertise, and may require significant sample preparation. Cryo-TEM images can contain increased background noise, though this can be mitigated by switching the buffer or the grid type2. Cryo-TEM imaging provides thorough information and direct visualization on a per-particle level of nanoparticles in their native state.

To learn if cryo-TEM is right for you, reach out now.
Cryo-TEM Dynamic Light Scattering (DLS) Analytical Ultracentrifugation Sedimentation Velocity (AUC) Size Exclusion Chromatography (SEC)
  • Direct visualization on a per-particle basis of nanoparticles in their native state.
  • Non-destructive, high throughput measurement of particle size.
  • Accurate analysis of full to empty capsids.
  • Provides information on size distribution and purity of nanoparticles.
  • Highly specialized equipment and expert sample preparation required
  • Cannot provide information on concentration.
  • Assumes particles are spherical and homogenous.
  • Low throughput, highly specialized equipment, time-consuming.
  • Requires large sample volume.
  • Unable to visualize blebs in lipid nanoparticles, or resolve intermediate capsids in AAV.

To learn if a cryo-TEM study is right for your project goals and your sample type, reach out to our team to schedule a meeting.

Menu Hex HoverMenu HexArrow pointing right to indicate exploring the Nano Imaging Services (NIS) difference.


  1. Markova N, Cairns S, Jankevics-Jones H, Kaszuba M, Caputo F, Parot J. Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques. Vaccines. 2022; 10(1):49. 
  2. Randy Crawford, Belma Dogdas, Edward Keough, R. Matthew Haas, Wickliffe Wepukhulu, Steven Krotzer, Paul A. Burke, Laura Sepp-Lorenzino, Ansuman Bagchi, Bonnie J. Howell, Analysis of lipid nanoparticles by Cryo-EM for characterizing siRNA delivery vehicles, International Journal of Pharmaceutics, Volume 403, Issues 1–2, 2011,  
  3. Amanda K. Werle,Thomas W. Powers, James F. Zobel, Caitlin N. Wappelhorst, Martin F. Jarrold, Nicholas A. Lyktey, Courtney D.K. Sloan, Andrew J. Wolf, Sharee Adams-Hall, Phoebe Baldus, Herbert A. Runnels, Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors, Molecular Therapy, Methods & Clinical Development, Volume 23, P254-262, 2021,   
  4. Xiujuan Jia, Yong Liu, Angela M. Wagner, Michelle Chen, Yuejie Zhao, Katelyn J. Smith, Dan Some, Andreas M. Abend, Justin Pennington, Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations, Journal of Chromatography B, Volume 1186, 2021, 
  5. Edwards, G. B., Muthurajan, U. M., Bowerman, S., & Luger, K. (2020). Analytical ultracentrifugation (AUC): An overview of the application of fluorescence and absorbance AUC to the study of biological macromolecules. Current Protocols in Molecular Biology, 133, e131. 
  6. Linde Schoenmaker, Dominik Witzigmann, Jayesh A. Kulkarni, Rein Verbeke, Gideon Kersten, Wim Jiskoot, Daan J.A. Crommelin, mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability,International Journal of Pharmaceutics, Volume 601,2021,

Infographic Available for Download

Fill out this form to download the infographic.

Understand the pros and cons of using cryo-EM, dynamic light scattering, analytical utracentrifugation, and size exclusion chromatography for nanoparticles.

NIS Mountain Hero
No items found.
No items found.