These are two different techniques which rely on two different modes of analysis. TEM records two-dimensional images of the object for comparison to a scale of measure. DLS measures very small particles in suspension using light scattering. These are two very different methods which are not comparable. Light-scattering methods require the use of known, measured standards for comparison. Differences in physical structure and dynamic volume must be taken into account. *Before using any analytical technique it is critical that you first take the time to learn HOW it works, what sample types are appropriate and how to interpret the output data.

Usually it’s the other way around with DLS results being larger than TEM. Many discussions on RG on the topic that you can search on. Has your TEM preparation method aggregated and agglomerated the protein sample? Some examples would be very helpful if you post these.

I think there may not be aggregation/agglomeration in my TEM samples, because I get uniformly dispersed particles in the TEM images. For DLS also I get single peaks. The only problem is that the particle sizes of TEM are way larger than DLS hydrodynamic sizes. It is really unexpected and I am still unable to figure out why. Despite repeating multiple times, I'm getting the same results. I will get a better look in the earlier RG articles once again. Thank you for the reply :) Alan F Rawle

The size difference between particles observed in DLS and TEM is common and likely results from the distinct measurement principles of each technique. DLS determines the hydrodynamic diameter, capturing not only the protein core but also the surrounding hydration layer or any loosely bound solvent molecules, which can make particles appear larger. In contrast, TEM measures the dry particle size directly in high vacuum, where hydration layers are absent.

Which TEM technique has you used? Samples for CryoTEM are vitrified, but TEM samples need the use of contrast agents and drying process that could modify some aggreggation property of your protein

Well, the problem with comparing these technique directly is that while one is a direct technique the other is indirect. DLS measures intensity fluctuations of scattered light, from which it gets particle diffusion, and from there you get particle size distributions. Moving from the initial signal to particle size measurements involves several matematical steps, so just from that you would already expect differences. Now, also think that in DLS you need ot input things like viscosity of the solvent, and temperature, which directly impact particle size. If the viscosity you are using is a bit off for the temperature at which the measurement is done, for instance, your particle size will change. In your specific case, if you are using a viscosity that is a bit too high, for instance, then the DLS-measured particle size will have an error towards smaller particle sizes. To add to this, some phenomena can affect particle diffusion (like for instance collective diffusion), also decreasing the apparent size when measured via DLS style techniques. Internal flows in your sample for instance can also make a DLS measurement yield smaller particle sizes that it would do if flows were not present.

Keep in mind that sometimes even surfactants in your liquid can change its viscosity and result in sinificant changes in article size as per DLS.