We developed metasurface-enhanced mid-infrared microscopy for living cells, where cells are cultured on a metasurface-bottomed microwell and are probed from the bottom using a confocal laser scanning microscope with a quantum cascade laser (QCL) as the source. The mid-infrared light interacts with the cells through the near-field of the plasmonic nanoantennas in the metasurface, and the reflected light is detected in the far field. Using this setup, we imaged fixed cells at different vibrational bands for the chemical imaging of different biomolecules, including proteins, lipids, and nucleic acids. Additionally, we have monitored the real-time adhesion and spreading of living cells on the metasurface with protein contrast.
Vertical nanostructures have been studied extensively and been reported to induce unique responses in cells, including the formation of clathrin coating and accumulation of actin filament which leads to cell membrane curvature around the nanostructures. In our previous works, we have demonstrated that arrays of plasmonic nanoantenna can be used as effective label free biosensors to study live cells when combined with IR spectroscopy in the reflection mode, known as metasurface enhanced IR spectroscopy (MEIRS). By fabricating these plasmonic metasurfaces on top of silica nanopillars, we demonstrate that we can expand the potential of MEIRS: increased protein IR absorption in the amide band and increased sensitivity of our biosensor to processes involving cellular interaction with vertical nanostructures.
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. 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). The application provides a novel tool to the drug discovery and fundamental cell biology research.
Plasmonic metasurface for surface-enhanced infrared absorption (SEIRA) spectroscopy is integrated with multi-well cell culture chambers for application as a cellular assay based on Fourier transform infrared (FTIR) spectroscopy. Live cells are grown directly on the metasurface, and the enhanced infrared absorption through metasurface plasmonic resonance is probed in reflection mode using a custom-built FTIR-coupled inverted infrared microscope. The application of the proposed device as a cellular assay is demonstrated through the measurement of cellular adhesion with different surface coatings, as well as cellular response to the stimulation of the protease-activated receptors (PARs).
Surface-enhanced infrared absorption (SEIRA) based on top-down fabricated nanostructures such as nanoantennas and metasurfaces has attracted much attention in recent years. These plasmonic resonant nanostructures can enhance the IR absorption signal of nearby molecules through its nearfield enhancement and have been shown to be able to detect adsorbed monolayers of proteins and lipids through their IR absorption spectra. Here, we demonstrate the continuous monitoring of cellular responses to stimuli using metasurface-enhanced infrared spectroscopy (MEIRS). A431 cells are seeded on a gold plasmonic metasurface fabricated on CaF2 substrate. Continuous monitoring is made possible by integrating the metasurface with a flow chamber, and the IR absorption spectra of the attached cells are measured in reflectance mode under continuous perfusion of cell culture medium. Scanning electron microscopy (SEM) revealed that the cells preferentially adhere to gold surfaces rather than CaF2 surfaces, suggesting that the IR signal measured through MEIRS is highly sensitive to the cells’ attachment and interaction with the gold metasurface. We have monitored the effect of methyl-beta-cyclodextrin, a cholesterol-depleting compound, on A431 cells. Principal component analysis highlighted the complex and subtle spectral changes of the cells.
Optical spectroscopy is a powerful technique that allows for label-free, noninvasive, and real time characterization of biomolecules. Compared with other optical techniques that relies on the shift of a single resonance, such as surface plasmon resonance (SPR) sensors and optical-grating-based cell assays, spectroscopic techniques can discriminate between different chemical species and are suited for analyzing often complex biological samples. Surface-enhanced infrared absorption (SEIRA) based on top-down fabricated substrates such as nanoantennas, nanoslits, and metasurfaces has been demonstrated as a versatile technique that can enhance the IR signal and characterize small amounts of adsorbed protein and lipid films. Here, we demonstrate the use metasurface-enhanced infrared reflection spectroscopy (MEIRS) to observe live cells cultured on top of the plasmonic metasurface. MEIRS has a penetration depth on the order of tens of nanometers, longer than surface-enhanced Raman spectroscopy (SERS) and yet shorter than attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. This makes MEIRS uniquely suited for probing the surface of a cell, to observe features such as protein expression in the cell membrane and cellular adhesion. This has important implications in the detection of cancer cells through spectroscopic cytology, as cancer cells differ from regular cells significantly in the expression of membrane proteins and adhesion molecules. In this work, we demonstrate the observation of cancer cell adhesion through IR spectroscopic mapping. Furthermore, we show the effect of different anticancer cocktails (doxorubicin, salinomycin, and their combination) on cancer cells, as observed by MEIRS.
In this talk I will discuss surface enhanced Raman scattering in silica microsphere resonators based on whispering gallery mode resonance. Recently silica microspheres have attracted attention as a novel substrate for surface enhanced Raman scattering. Whispering gallery mode resonance has been identified as a major enhancement mechanism, along with other effects such as photonic nanojets. In most of the previous experiments, however, free space pumping of the microsphere has been used, which has low efficiency in coupling to the whispering gallery modes. In our approach, we use a tapered fiber coupler for a highly efficient coupling to the whispering gallery modes. Coupling to the microresonator is monitored using a tunable laser. We observe both pump enhancement and Purcell enhancement in the microresonator. Since the linewidth of the whispering gallery modes is much smaller than that of the Raman peaks, sharp peaks corresponding to the whispering gallery modes are overlaid on top of the Raman spectrum of the bulk material. To demonstrate the system’s potential for Raman analysis, I will present the whispering gallery mode surface enhanced Raman spectrum of rhodamine 6G thin film coated on a microsphere resonator.
Ultrahigh-quality whispering gallery mode optical microresonators have been studied for their use as highly sensitive sensors. In this talk, we discuss the use of microsphere microresonators in Raman spectroscopy for interrogating particles adhered to the surface of the resonator. An external cavity diode laser is tuned to a resonant high-Q mode and the circulating optical field experiences a large buildup, resulting in enhanced Raman scattering. Here we present studies of Raman scattering spectroscopy of single particles. Raman sensing with different Q's is discussed.
The idea of creating photonics tools for sensing, imaging and material characterization has long been pursued and many achievements have been made. Approaching the level of solutions provided by nature however is hindered by routine choice of materials. To this end recent years have witnessed a great effort to engineer mechanically flexible photonic devices using polymer substrates. On the other hand, biodegradability and biocompatibility still remains to be incorporated. Hence biomimetics holds the key to overcome the limitations of traditional materials in photonics design. Natural proteins such as sucker ring teeth (SRT) and silk for instance have remarkable mechanical and optical properties that exceed the endeavors of most synthetic and natural polymers. Here we demonstrate for the first time, toroidal whispering gallery mode resonators (WGMR) fabricated entirely from protein structures such as SRT of Loligo vulgaris (European squid) and silk from Bombyx mori. We provide here complete optical and material characterization of proteinaceous WGMRs, revealing high quality factors in microscale and enhancement of Raman signatures by a microcavity. We also present a most simple application of a WGMR as a natural protein add-drop filter, made of SRT protein. Our work shows that with protein-based materials, optical, mechanical and thermal properties can be devised at the molecular level and it lays the groundwork for future eco-friendly, flexible photonics device design.
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