Hi all,
In my experiment the liposome size is obtained from DLS and SEM measurements for the same formulation do not match. Why is that? Any reference links will be helpful. Thanks.
Hello Sarbani,
DLS (Dynamic Light scattering technique) is well versed used for studying hydrodynamic properties of the material. So the average size what you see in DLS is the hydrodynamic radius of the material. While in Scanning Electron microscope we dry the sample and view it, depending on the resolution of the SEM. This means, the size study given by DLS technique and SEM is not similar ever. Sample preparation is there since both the techniques have a different way of characterisation.
Note :- In DLS technique we use a 700 nm (red) laser and the event study is on the basis of scattering and mobility of material. In SEM we use an electron beam and our study is done on back scattering or secondary scattering events etc. from the material .
Hope this helps u in understanding more...
Mahesh
not necessary. surely it vary. in my experience. What you are observe in SEM is not 100% similar with DLS. there must be fluctuation in that value due to sample preparation for both the analysis
Hello Sarbani,
DLS (Dynamic Light scattering technique) is well versed used for studying hydrodynamic properties of the material. So the average size what you see in DLS is the hydrodynamic radius of the material. While in Scanning Electron microscope we dry the sample and view it, depending on the resolution of the SEM. This means, the size study given by DLS technique and SEM is not similar ever. Sample preparation is there since both the techniques have a different way of characterisation.
Note :- In DLS technique we use a 700 nm (red) laser and the event study is on the basis of scattering and mobility of material. In SEM we use an electron beam and our study is done on back scattering or secondary scattering events etc. from the material .
Hope this helps u in understanding more...
Mahesh
Anyway, depending the nature of your sample in hard nanomaterials the average DLS size by number, is always most like the SEM observations.
I think medium may be one reason for size difference, if your materials is water absorb-able because in DLS your sample remains hydrated while in SEM sample is in dry state.
Adding to above answers, I wish to add SEM is not an ideal way when one measures size of the vesicles, as while drying the molecules changes its interaction with neighboring molecules due to lack of hydration hence the size varies from the size when in any medium.
The observations from DLS and SEM vary as explained by Mahesh Samant
Using DLS one can not confirm about the size and shape of the particle. Some times in case of aggregated forms, DLS data shows more larger particle size , i.e., Hydrodynamic diameter. But SEM confirms you the exact structure and shape of the particle . Results may vary. Better you should go with the SEM or TEM data or may AFM.
The only way to get a true figure for liposomes is to freeze fracture them and then do TEM on the replicas as most EM prep techniques involve some sort of drying and fixation techniques. Freez fracture kit is quite rare these days though.
One of the most artifactual-free TEM technique is Cryo-TEM, due to way used to prepare the sample. Classical TEM can be useful, but a lot of images has to be obtained.
On the other hand, as many colleagues said before, DLS measures the hydrodynamic diameter. This distance can become different from that corresponding to the "real" liposomes if these vesicles are in the SUVs range (when the hydration layer is not small compared with the vesicle size).
Finally, keep in mind the meaning of the size distributions obtained by DLS and electronic microscopy. DLS devices can provide number, volume (or mass) and area distributions of the analyzed samples. The interpretation of the data depends on what are you interested to know, and all distributions give important and complementary information. Images obtained by optical or electronic devices provide number distributions but, of course, you can obtain the other distributions from the original data.
In DLS method, measured size of particle is dependent to power six of particle diameter. and it measured hydrodynamic size of particles.
There isn't a direct relation between measured size by these two methods, but in most of measurements, the size which measured by DLS is larger that SEM method.
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