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Analyzing Mixed Nanoplastics for Environmental Studies


Microplastics, defined as plastic fragments less than 5 mm in length, are classified as primary or secondary depending on if the fragments were already less than 5 mm before entering the environment. In 2014, between 15 and 51 trillion pieces of microplastics (weighing between 93,000 and 236,000 metric tons) are estimated to be present in the world’s oceans.1

Nanoplastics aren’t as well defined, but they are usually plastic fragments less than 100 or 1000 nm in size and can be classified similarly as primary or secondary. While their impacts are still unknown, nanoplastics are thought to be more of a risk to environmental and human health since their small size allows them to cross cellular membranes.

Conventional analysis techniques

Microplastics are generally studied and identified using either FTIR or Raman spectroscopy. When studying particles greater than about 10 microns in size, FTIR is generally preferred because it is considered to be an easier technique. For smaller particles, Raman spectroscopy is used for its higher spatial resolution despite its many weaknesses such as the background fluorescence signal, low signal, or potential damage to particles from the use of laser excitation.2

Photo-induced force microscopy and spectroscopy

While the presence of nanoplastics is certain, the extent of their concentration is unknown due to the lack of analytical techniques for nanoscale chemical analysis. However, the advent of photo-induced force microscopy (PiFM) and photo-induced force infrared (PiF-IR) spectroscopy provided techniques perfectly suited for such characterizations.

IR PiFM is an excellent tool for analyzing nanoplastics for various reasons: (1) its capability to categorize both the size and the chemical identity for particles down to about 5 nm in size; (2) its capability to identify both organic and inorganic nanoparticles; (3) its capability to “see through” thin layers of biological contaminants to identify underlying nanoparticles; (4) its excellent sensitivity without any concern for fluorescence interference; (5) its non-contact and non-damaging measurement capabilities; and (6) its simple sample preparation requirements.

Sample analysis

As a demonstration of PiFM’s capabilities, we analyze a sample that consists of four different nanoparticles: PTFE, PMMA, PS, and gold nanoparticles. The nanoparticles are drop cast on a poly-l-lysine substrate. This sample should serve as a reasonable proxy for the complex and varied environmental samples that one might study.

A Vista One IR microscope was used to make an initial AFM image of the sample’s surface. Figure 1 shows the topography in both a 2D grayscale and 3D format. With standard microscopy techniques, there would be no way to identify the chemical identity of such small particles.

Figure 1. Topography and 3D rendering of the sample surface with four different types of nanoparticles. Conventional microscopy techniques can’t identify chemicals at this scale.

The Vista One was then used to take PiF-IR spectra on different particles in the AFM image. Three unique spectra were obtained, and figure 2 shows the locations they were acquired from. These spectra are compared to bulk FTIR spectra for each of the materials, which we gleaned from the internet. The PiF-IR spectra from individual particles match the bulk FTIR spectra closely enough that they can be used to chemically identify the different particles.

Figure 2. PiF-IR spectra acquired on different particles agree well with the bulk FTIR spectra for each of the materials allowing us to chemically identify each of the nanoparticles.

With the material identifications made, PiFM images can be used to create chemical distribution maps. The prominent vibrational bands associated with the different particles are used to acquire PiFM images that highlight each type of nanoparticle. Images at 1732 cm−1, 1158 cm−1, and 1493 cm−1 highlight the PMMA, PTFE, and PS nanoparticles, respectively.

Imaging the gold nanoparticles is more challenging because they have no IR active band. However, the tip-enhanced field will be greater when the tip is over the gold particles, leading to a higher background PiFM signal. Therefore, we can use a wavenumber such as 1800 cm−1, where there is no other vibrational band for other particles, to highlight the gold nanoparticles. Interestingly, PTFE seems to be highlighted as well at 1800 cm−1 due to higher background signals associated with the PTFE particles. This may be due to slightly different mechanical tip-sample interactions when the tip is over PTFE particles.

With that done, we now have four PiFM chemical concentration maps to show the locations of all four types of nanoparticles. The complete set of images is shown in figure 3.

Figure 3. PiFM images at 1732 cm−1, 1158 cm−1, and 1493 cm−1 uniquely highlight the PMMA, PTFE, and PS nanoparticles, respectively. A PiFM image at 1800 cm−1 highlights the gold nanoparticles along with PTFE.

Next, we can use the AFM capabilities of Vista One to measure the actual sizes of the particles. In Figure 4, we see the cross-sections of 4 representative particles. In AFM, the tip’s radius of curvature will dilate the lateral size of the particle; for spherical particles, the height of the particle will be a good measure of the particle size. Therefore, using height measurements, we see that the sizes of PTFE, PMMA, PS, and gold nanoparticles are about 190 nm, 40 nm, 70 nm, and 10 nm in size, respectively.

Figure 4. The sizes of the PTFE, PMMA, PS, and gold nanoparticles are about 190 nm, 40 nm, 70 nm, and 10 nm, respectively.

To pull this dataset together into a single image, the PiFM chemical maps can be composited and overlaid on the 3D AFM topography. Figure 5 shows the result with different colors for each type of nanoparticle: green for gold and PTFE, red for PS, and blue for PMMA. An image like this provides an easy-to-understand view of the sample and makes it clear that one could not use size or other clues from the topography alone to classify the nanoparticles. This demonstrates the invaluable features PiFM and PiF-IR bring to analyzing complex nanoscale systems.

Figure 5. A composite of three PiFM images is overlaid on the 3D topography to highlight different nanoparticles with different colors.


In summary, PiFM and PiF-IR can chemically identify and measure the sizes of nanoplastic particles along with other inorganic and biological nanoparticles for environmental forensics with unprecedented spatial resolution.3 The sample studied here serves as a substitute for the types of complex systems that would be seen in environmental samples, demonstrating that PiFM and PiF-IR fill a necessary role in modern microscopy.


  1. Sebille, Erik van. “Far more microplastics floating in oceans than thought”. The Conversation. Retrieved 2021-07-07.
  2. C. F. Araujo et al., Identification of microplastics using Raman spectroscopy: Latest developments and future prospects, Water Research 142, 426 (2018)
  3. For more application notes on inorganics and biological samples, visit

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