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 [1]
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 [2]:
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-enabled inverted reflected-light infrared absorption microscopy (MIRIAM), 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. For hydrated fixed cells, hyperspectral data cubes were sequentially obtained as two-dimensional (2D) images at DFs, enabling the imaging of the cellular organelles (nuclei, cytoplasm, and lipid droplets) distinguished by the MIR vibrations of their predominant molecules – nucleic acids, proteins, and lipids, respectively. Additionally, the MIR “movies” of living cells in culture medium were acquired as time-lapse images at one or several wavenumbers, enabling the observation of dynamic cellular processes such as cell spreading and locomotion. 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. Home-built QCL laser scanning microscope. The scanning function is implemented with a programmable high-precision microscope motorized stage.
Figure 4. MIR versus stained cell images
Video 1. The video of cell adhesion captured by the QCL microscope at 1502invcm.
Figure 5. Bright field, Phase contrast, MIRIAM at amide II and at 12 C=O ester band images of 3T3-L1 cells on the metasurface. White dashed curve indicates the boundary of an undifferentiated fibroblast cells, barely visible in bright field image. [3]
3. Integrating metasurfaces with nanostructured substrates – 3D metasurfaces [4]
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.
Related publications:
[1] S. H. Huang, G. Sartorello, PT. Shen, C. Xu, O. Elemento, and G. Shvets, Metasurface-enhanced infrared spectroscopy in multiwell format for real-time assaying of live cells, Lab on a Chip 23, 2228 (2023).
[2] S. H. Huang, PT. Shen, A. Mahalanabish, G. Sartorello, J. Li, X. Liu, and G. Shvets, Mid-infrared chemical imaging of living cells enabled by plasmonic metasurfaces, bioRxiv.
[3] S. H. Huang, D. Tulegenov, and G. Shvets, Combining quantum cascade lasers and plasmonic metasurfaces to monitor de novo lipogenesis with vibrational contrast microscopy, Nanophotonics (2025).
[4] A. Mahalanabish, S.H. Huang, D. Tulegenov, and G. Shvets, Infrared Spectroscopy of Live Cells Using High-Aspect-Ratio Metal-on-Dielectric Metasurfaces, Nano Lett. 24, 11607–11614 (2024).