# Microplastic Pollution: Chemical Characterization and Impact on Wildlife
## 2 The Chemical and Physical Characterization of Microplastics
### Electron Microscopy  
An additional technique commonly used to characterize the size, shape, and chemical composition of microplastics is Scanning Electron Microscopy (SEM). This technique is used to image and measure objects with different diameters ranging from millimeters to nanometers in size [50]. The SEM technique is capable of nanometer spatial resolution because the deBroglie wavelength of the electron beam used to probe a sample is far less than optical wavelengths, and as such, the optical diffraction limitation is overcome. The electron beams used by scanning electron microscopes (SEMs) can produce images of  
Figure 2: (**A**) Optical microscope images of microplastic particles: (**a**) Polyethylene (PE) particle; (**b**) polyethylene terephthalate (PET) particle; (**c**) polypropylene (PP) particle; (**d**) polyvinyl chloride (PVC) particle. (**B**) Microplastics of six different polymer types, dyed with Nile Red (1000 μg\(\cdot\)mL\({}^{-1}\) for 30 min). Figures reproduced from [PERSON] et al. [38] and [PERSON] et al. [46] with permission of publishers. samples with a resolution of just a few nanometers. Thus, SEM helps observe tiny particles that an optical microscope may miss [51]. A schematic of an SEM is illustrated in Figure 3A. Within the device, the electron source's filament emits electrons, which are then collimated into a beam. The electron column's set of lenses then focuses the beam on the sample surface [52]. SEM offers outstanding depth of field, high resolution, and strong sensitivity over a wide range of materials, overcoming many of the traditional limits of light microscopy [53]. The electron optics of an SEM are designed to produce a tiny volume electron probe at the specimen focal plane by demagnifying the tiniest virtual cross-section of the electron beams close to the cathode. A voltage of 0.1 to 50 keV between the cathode and anode accelerates the electrons released from thermionic, Schottky, or field-emission cathodes. Then, the electron beam is swept across the sample while recording the electrons that bounce back (scattered) or are transmitted. SEM can be combined with a technique known as energy-dispersive X-ray analysis (EDX) in which X-rays are produced during the samples' irradiation with the electron beam and the signal produced provides chemical information. EDX analysis works because the electron probe beam hits the inner core-shell electrons of an atom, knocking off an electron from the core-shell. This creates an electron 'hole' or a vacancy a higher energy electron will rapidly fill. As the electron moves from the outer higher-energy to the inner lower-energy shell of the atom, the energy difference must be released to the surroundings, in the form of an X-ray. The energy of this X-ray is unique to the specific element. Thus, collecting these X-rays and analyzing them allows elemental analysis to be performed upon the sample. Figure 3B illustrates SEM images of fiber particles and the EDX spectra which provide evidence for a variety of particle compositions for suspect microplastic particles isolated  
Figure 3: (**A**) Schematic of an SEM instrument [54]. (**B**) SEM images (**a1–d1**) and EDX spectra (**a2–d2**) from suspect microplastic particles isolated from marine organisms. SEM with EDX allows investigators the ability to exclude particles from being microplastics, as is case of object (**d1**), which appears to be silica [51]. (**C**) SEM images from microplastic (**A–D**); SEM images of microplastic debris extracted from the stomach contents of _Bursatella leachii_ specimens [55]. Figures have been reproduced with permission of publishers. from marine organisms. The presence of an abundance of carbon and oxygen, along with fluorine, may suggest synthetic origins. In the study of [PERSON] et al. (2018), the authors used SEM with EDX to screen particles collected from marine organisms for shape and size along with chemical composition.