Infrared spectroscopy has emerged as the leading tool for biosensing because of its inherent chemical specificity and the capability for label-free, noninvasive, and real-time detection of constituent biological molecules. Applied to cells, the amount of proteins, lipids, nucleic acids and carbohydrates can be identified through infrared spectroscopy and used to identify different cell types, as well as monitor dynamic changes in the cells. Nanophotonics has played a crucial role for pushing the sensitivity limits of traditional far-field spectroscopy by using resonant nanostructures to focus the incident light into nanoscale hot-spots of the electromagnetic field, greatly enhancing light– matter interaction. Metasurfaces composed of regular arrangements of such resonators allows us to tailor this nanoscale light spectrally. Current areas of ongoing research in this area include:
1. Monitoring cellular response to stimuli through metasurface-enhanced infrared spectroscopy
Our group have used metasurface-enhanced infrared spectroscopy (MEIRS) to monitor various cellular dynamics, including cell adhesion, cholesterol depletion, activation of signaling receptor, and cell’s response to chemotherapeutics. We are interested in developing MEIRS as a cellular assay technique that can be used to study cell-drug interactions and cellular metabolism.
Figure 1. Plasmonic metasurface integrated with 2 × 8 well cell culture chamber.
Figure 2. MEIRS observation of cellular response to cholesterol depletion through methyl-beta cyclodextrin (MBCD). Different temporal response can be seen through protein absorption, lipid absorption, and plasmonic resonance shift (i.e. refractive index change).
2. Metasurface-based cell imaging system:
Live-cell mid-infrared (MIR) imaging has always been challenging because of the absorptive nature of water. However, there is a strong drive to image this spectroscopic window – to see the protein and lipid vibrations directly without the help of dyes. Though the dyes are convenient for imaging, they interfere with the biological functions of live cells. Some popular techniques such as stimulated Raman scattering (SRS) or coherent anti-Stokes Raman scattering (CARS) microscopy are capable of chemically imaging the live cells without labelling, but the high optical power required for the nonlinear scattering of light is still phototoxic. IR chemical imaging, on the other hand, typically require much smaller optical power, so the phototoxicity is greatly reduced.
In the past two decades, people have relied on attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopic imaging to probe such systems to reduce the infrared penetration depth to a few microns. In our previous works, we found a way to further restrict the penetration to a hundred nanometers with plasmonic nanoantennas, also known as the metasurfaces. We named the technique – metasurface-enhanced infrared reflection spectroscopy (MEIRS), and used it for either label-free spectroscopy or imaging. We had demonstrated MEIRS in various live-cell drug dynamics studies, including trypsin, cholesterol depleting agents, and chemotherapeutics, of live cells enclosed in microfluidics chambers. With the recent advancement of commercial mid-infrared quantum cascade laser (QCL), we now have a unique opportunity to acquire high-quality single-cell resolution metasurface-enhanced infrared reflection chemical imaging (MIRCI), which reveals the important protein information in real time. We built an inverted QCL microscope setup and cultured the cells on a cell-culture multiwell plate. The bottom of the multiwells is made of infrared-transparent window and with metasurface fabricated on. In this work, we demonstrated two proofs of concept of MIRCI on both fixed cells in water (single-cell resolution and spectroscopy) and live cells (capturing cell adhesion process). We also found the laser power in use could be as low as 10 µW. The power is only 1/1000 of the laser power used in Raman-based techniques. The application provides a novel tool to the drug discovery and fundamental cell biology research.
Figure 3. (a) Home-built QCL laser scanning microscope. The scanning function is implemented with a programmable high-precision microscope motorized stage. (b) SEM image of fixed cells on the Π metasurfaces.
Figure 4. MIR single-cell spectroscopy and chemical imaging: (a) Select a cell with phase contrast microscopy. (b) Perform hyperspectral imaging. (c) Position the stage at the selected cell and no-cell coordinates and perform a frequency sweep at each coordinate.
Figure 5. Chemical imaging(Amide II) snapshots of live cell adhesion form suspension state.
Video 1. The video of cell adhesion captured by the QCL microscope at Amide II.
3. Integrating metasurfaces with nanostructured substrates – 3D metasurfaces
Cellular adhesion to surfaces strongly depends on the topography of the surface. Surfaces with nanotopographic features like the one shown in figure 6(a) incite unique chemical and physical responses in cells. One of the main physical responses is cell membrane curvature, which comes about from a process called clathrin mediated endocytosis (CME). Endocytosis incites cell membrane wrapping around such structures by closely following the nanotopography on the surface, as shown in figure 6(c) below. Endocytosis forces cells to attach more closely to the resonant metasurface allowing for increase in overlap of the nearfields with the cell volume, which in turn leads to increased molecular IR signal observed in the far field metasurface spectra (figure 7(a)). Since the attachment process itself is different from that on flat surfaces (like in 2D metasurfaces), we also observe a difference in the secondary structure of the proteins being sensed optically (figure 7(b)).
Figure 6: (a) Schematic of the fabricated 3D metasurface. (b) Schematic showing optical processes responsible for the measured cellular spectra. (c) SEM image showing A431 cell grown on fabricated 3D metasurface sample. (d) FIB-SEM image showing a cross-sectional view of the cell attached to the nanopillar structure.
Figure 7: (a) Comparison of absorbance signal for A431 cells grown on 3D metasurfaces vs that on 2D metasurface. (b) Second derivative spectra obtained from the absorbance, showing secondary protein structure.
Shen, P. T., Huang, S. H., Huang, Z., Wilson, J. J. & Shvets, G. Probing the Drug Dynamics of Chemotherapeutics Using Metasurface-Enhanced Infrared Reflection Spectroscopy of Live Cells. Cells 11, 1600 (2022).
Huang, S. H., Li, J., Fan, Z., Delgado, R. & Shvets, G. Monitoring the effects of chemical stimuli on live cells with metasurface-enhanced infrared reflection spectroscopy. Lab Chip 21, 3991–4004 (2021).
Kelp, G. et al. Infrared spectroscopy of live cells from a flowing solution using electrically-biased plasmonic metasurfaces. Lab Chip 20, 2136–2153 (2020).
Kelp, G. et al. Application of metasurface-enhanced infra-red spectroscopy to distinguish between normal and cancerous cell types. Analyst 144, 1115–1127 (2019).
Wu, C. et al. Fano-resonant asymmetric metamaterials for ultrasensitive spectroscopy and identification of molecular monolayers. Nat. Mater. 11, 69–75 (2011).