6.4.3　Morphology

Microscopes，including transmission electron microscope（TEM），scanning electron microscope（SEM），and atomic force microscope（AFM），are used to measure the morphology of polymer nano-drug carriers.

TEM is the most commonly used tool to measure the morphological structure of polymer nano-drug carriers.It can image objects of several nanometers to micrometers with high resolution.It also provides nanostructure and elemental information through different imaging modes（e.g.，freezing mode and scanning transmission mode）.During testing of TEM，the electron beams pass through the sample and focus on the object lens.The intensity and angle of electron beams vary with the differences in element，density，and thickness of NPs.As a result，different morphology and contrast in the image reflect the different structures and materials of the NPs.

Staining is usually used to improve the contrast of polymer NPs under TEM.There are two staining methods—positive staining and negative staining.In positive staining，the NPs are stained by high contrast materials，so that the NPs show darker appearances in the TEM image.For example，uranyl acetate can be used to stain proteins and other polymer NPs containing amino groups.Negative staining improves the contrast of the sample in the imaging by increasing the contrast of the background.The negatively stained sample displays a dark background in the TEM image，while the sample is bright due to the less stain.The staining method can improve the contrast of some polymer NPs as well as provide the microstructures of the particles.

Cryo-TEM is the direct method to observe polymer NPs in the solution phase.It uses electron beams to image the NPs frozen in the liquid（Figure 6-8（a））.Three scientists who developed Cryo-TEM technology to measure the high-resolution structure of biological macromolecules were awarded the 2017 Nobel Prize in Chemistry，demonstrating the critical role of Cryo-TEM in structural measurement（see extended reading 3）.

Besides，there are other modes for TEM measurement.For example，high-resolution TEM（HRTEM）can image the lattice between atoms in nanomaterials，which enables us to characterize inorganic/polymer composite NPs.In addition，the combination of computerized tomography imaging technology and the mathematical model of image analysis can reconstruct the three-dimensional structure image of the NPs，so that we will have a more intuitive and accurate understanding of the morphology of the NPs with the threedimensional structure.Furthermore，the TEM energy-dispersive X-ray spectroscopy（EDXS），electron energy loss spectroscopy（EELS）and other technologies enable us to measure the distribution of elements in NPs.(https://www.daowen.com)

In short，the high-resolution and multifunctional measurement capabilities of TEM have made it an indispensable technique in the measurement of nano-drug carriers.

SEM is a technology that visualizes the surface topography of NPs using electron beams.Generally，the resolution of SEM is less than TEM.Moreover，materials with poor conductivity need to be covered with a thin gold layer on the surface of the sample to increase the conductivity.The advantage of SEM compared with TEM is that it can load more samples at one time.Considering that samples need to be vacuumed during the test process of SEM and TEM，SEM can check more samples than TEM in a certain period.In addition，SEM provides more information about the surface structure of NPs.Therefore，SEM is more suitable for the measurement of the external morphology of large-sized conductive materials or inorganic/polymer composite nanomaterials.

AFM can perform high-resolution three-dimensional imaging of the surface structure of NPs.It interacts with the particle surface through a cantilever and a fine probe connected to the cantilever to collect corresponding imaging information.Except for the morphology of NPs，AFM provides additional information such as particle height，softness，and hardness，which are of great significance for the analysis of two-dimensional polymer nanomaterials such as ring，sheet，and dish.Moreover，surface modification or spraying is not required during the sampling of AFM.For NPs with low contrast in TEM images，it also provides accurate morphology.Due to these advantages，AFM technique becomes an important technique for the measurement of the morphology of polymer NPs.