Dual-wavelength metalens enables Epi-fluorescence detection from single molecules.

Single molecule fluorescence spectroscopy is at the heart of molecular biophysics research and the most sensitive biosensing assays. The growing demand for precision medicine and environmental monitoring requires the creation of miniaturized and portable sensing platforms. However, the need for highly sophisticated objective lenses has precluded the development of single molecule detection systems for truly portable devices. Here, we propose a dielectric metalens device of submicrometer thickness to excite and collect light from fluorescent molecules instead of an objective lens. The high numerical aperture, high focusing efficiency, and dual-wavelength operation of the metalens enable the implementation of fluorescence correlation spectroscopy with a single Alexa 647 molecule in the focal volume. Moreover, the metalens enables real-time monitoring of individual fluorescent nanoparticle transitions and identification of hydrodynamic diameters ranging from a few to hundreds of nanometers. This advancement in sensitivity extends the application of the metalens technology to ultracompact single-molecule sensors.

Dual-wavelength UV-visible metalens for multispectral photoacoustic microscopy: A simulation study.

Photoacoustic microscopy is advancing with research on utilizing ultraviolet and visible light. Dual-wavelength approaches are sought for observing DNA/RNA- and vascular-related disorders. However, the availability of high numerical aperture lenses covering both ultraviolet and visible wavelengths is severely limited due to challenges such as chromatic aberration in the optics. Herein, we present a groundbreaking proposal as a pioneering simulation study for incorporating multilayer metalenses into ultraviolet-visible photoacoustic microscopy. The proposed metalens has a thickness of 1.4 µm and high numerical aperture of 0.8. By arranging cylindrical hafnium oxide nanopillars, we design an achromatic transmissive lens for 266 and 532 nm wavelengths. The metalens achieves a diffraction-limited focal spot, surpassing commercially available objective lenses. Through three-dimensional photoacoustic simulation, we demonstrate high-resolution imaging with superior endogenous contrast of targets with ultraviolet and visible optical absorption bands. This metalens will open new possibilities for downsized multispectral photoacoustic microscopy in clinical and preclinical applications.

Bright-Field and Edge-Enhanced Imaging Using an Electrically Tunable Dual-Mode Metalens.

The imaging of microscopic biological samples faces numerous difficulties due to their small feature sizes and low-amplitude contrast. Metalenses have shown great promise in bioimaging as they have access to the complete complex information, which, alongside their extremely small and compact footprint and potential to integrate multiple functionalities into a single device, allow for miniaturized microscopy with exceptional features. Here, we design and experimentally realize a dual-mode metalens integrated with a liquid crystal cell that can be electrically switched between bright-field and edge-enhanced imaging on the millisecond scale. We combine the concepts of geometric and propagation phase to design the dual-mode metalens and physically encode the required phase profiles using hydrogenated amorphous silicon for operation at visible wavelengths. The two distinct metalens phase profiles include (1) a conventional hyperbolic metalens for bright-field imaging and (2) a spiral metalens with a topological charge of +1 for edge-enhanced imaging. We demonstrate the focusing and vortex generation ability of the metalens under different states of circular polarization and prove its use for biological imaging. This work proves a method for in vivo observation and monitoring of the cell response and drug screening within a compact form factor.

Hyperlens for capturing sub-diffraction nanoscale single molecule dynamics.

Hyperlenses offer an appealing opportunity to unlock bioimaging beyond the diffraction limit with conventional optics. Mapping hidden nanoscale spatiotemporal heterogeneities of lipid interactions in live cell membrane structures has been accessible only using optical super-resolution techniques. Here, we employ a spherical gold/silicon multilayered hyperlens that enables sub-diffraction fluorescence correlation spectroscopy at 635 nm excitation wavelength. The proposed hyperlens enables nanoscale focusing of a Gaussian diffraction-limited beam below 40 nm. Despite the pronounced propagation losses, we quantify energy localization in the hyperlens inner surface to determine fluorescence correlation spectroscopy (FCS) feasibility depending on hyperlens resolution and sub-diffraction field of view. We simulate the diffusion FCS correlation function and demonstrate the reduction of diffusion time of fluorescent molecules up to nearly 2 orders of magnitude as compared to free space excitation. We show that the hyperlens can effectively distinguish nanoscale transient trapping sites in simulated 2D lipid diffusion in cell membranes. Altogether, versatile and fabricable hyperlens platforms display pertinent applicability for the enhanced spatiotemporal resolution to reveal nanoscale biological dynamics of single molecules.

Metasurfaces-Driven Hyperspectral Imaging via Multiplexed Plasmonic Resonance Energy Transfer.

Obtaining single-molecular-level fingerprints of biomolecules and electron-transfer dynamic imaging in living cells are critically demanded in postgenomic life sciences and medicine. However, the possible solution called plasmonic resonance energy transfer (PRET) spectroscopy remains challenging due to the fixed scattering spectrum of a plasmonic nanoparticle and limited multiplexing. Here, multiplexed metasurfaces-driven PRET hyperspectral imaging, to probe biological light-matter interactions, is reported. Pixelated metasurfaces with engineered scattering spectra are first designed over the entire visible range by the precision nanoengineering of gap plasmon and grating effects of metasurface clusters. Pixelated metasurfaces are created and their full dark-field coloration is optically characterized with visible color palettes and high-resolution color printings of the art pieces. Furthermore, three different biomolecules (i.e., chlorophyll a, chlorophyll b, and cytochrome c) are applied on metasurfaces for color palettes to obtain selective molecular fingerprint imaging due to the unique biological light-matter interactions with application-specific biomedical metasurfaces. This metasurface-driven PRET hyperspectral imaging will open up a new path for multiplexed real-time molecular sensing and imaging methods.

Ultraviolet Nanophotonics Enables Autofluorescence Correlation Spectroscopy on Label-Free Proteins with a Single Tryptophan.

Using the ultraviolet autofluorescence of tryptophan amino acids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labeling. However, the low autofluorescence signals have so far limited the UV detection to large proteins containing several tens of tryptophan residues. This limit is not compatible with the vast majority of proteins which contain only a few tryptophans. Here we push the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Our results show how the combination of nanophotonic plasmonic antennas, antioxidants, and background reduction techniques can improve the signal-to-background ratio by over an order of magnitude and enable UV-FCS on thermonuclease proteins with a single tryptophan residue. This sensitivity breakthrough unlocks the applicability of UV-FCS technique to a broad library of label-free proteins.

Ultraviolet optical horn antennas for label-free detection of single proteins.

Single-molecule fluorescence techniques have revolutionized our ability to study proteins. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with other species. To avoid the need for a fluorescent label, the intrinsic autofluorescence of proteins in the ultraviolet offers the benefits of fluorescence techniques without introducing the labelling drawbacks. Unfortunately, the low autofluorescence brightness of proteins has greatly challenged single molecule detection so far. Here we introduce optical horn antennas, a dedicated nanophotonic platform enabling the label-free detection of single proteins in the UV. This design combines fluorescence plasmonic enhancement, efficient collection up to 85° angle and background screening. We detect the UV autofluorescence from immobilized and diffusing single proteins, and monitor protein unfolding and dissociation upon denaturation. Optical horn antennas open up a unique and promising form of fluorescence spectroscopy to investigate single proteins in their native states in real time.

Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins.

The poor photostability and low brightness of protein autofluorescence have been major limitations preventing the detection of label-free proteins at the single-molecule level. Overcoming these issues, we report here a strategy to promote the photostability of proteins and use their natural tryptophan autofluorescence in the ultraviolet (UV) for fluorescence correlation spectroscopy (FCS). Combining enzymatic oxygen scavengers with antioxidants and triplet-state quenchers greatly promotes the protein photostability, reduces the photobleaching probability, and improves the net autofluorescence detection rate. Our results show that the underlying photochemical concepts initially derived for organic visible fluorescent dyes are quite general. Using this approach, we achieved UV fluorescence correlation spectroscopy on label-free streptavidin proteins containing only 24 tryptophan residues, 6.5× fewer than the current state-of-the-art. This strategy greatly extends the possibility of detecting single label-free proteins with the versatility of single-molecule fluorescence without requiring the presence of a potentially disturbing external fluorescent marker. It also opens new perspectives to improve the UV durability of organic devices.

Zero-mode waveguides can be made better: fluorescence enhancement with rectangular aluminum nanoapertures from the visible to the deep ultraviolet.

Nanoapertures milled in metallic films called zero-mode waveguides (ZMWs) overcome the limitations of classical confocal microscopes by enabling single molecule analysis at micromolar concentrations with improved fluorescence brightness. While the ZMWs have found many applications in single molecule fluorescence studies, their shape has been mainly limited to be circular. Owing to the large parameter space to explore and the lack of guidelines, earlier attempts using more elaborate shapes have led to unclear conclusions whether or not the performance was improved as compared to a circular ZMW. Here, we comparatively analyze the performance of rectangular-shaped nanoapertures milled in aluminum to enhance the fluorescence emission rate of single molecules from the near infrared to the deep ultraviolet. Our new design is based on rational principles taking maximum advantage of the laser linear polarization. While the long edge of the nanorectangle is set to meet the cut-off size for the propagation of light into the nanoaperture, the short edge is reduced to 30 nm to accelerate the photodynamics while maintaining bright fluorescence rates. Our results show that both in the red and in the ultraviolet, the nanorectangles provide 50% brighter photon count rates as compared to the best performing circular ZMWs and achieve fluorescence lifetimes shorter than 300 ps. These findings can be readily used to improve the performance of ZMWs, especially for fast biomolecular dynamics, bright single-photon sources, and ultraviolet plasmonics.

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides.

Single molecule detection provides detailed information about molecular structures and functions but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labeling. However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguide nanoapertures enable the observation of the UV fluorescence of single label-free ß-galactosidase proteins with increased brightness, microsecond transit times, and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient, and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. Although the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.

Preventing Aluminum Photocorrosion for Ultraviolet Plasmonics.

Aluminum can sustain plasmonic resonances down into the ultraviolet (UV) range to promote surface-enhanced spectroscopy and catalysis. Despite its natural alumina passivating layer, we find here that under 266 nm pulsed UV illumination, aluminum can undergo a dramatic photocorrosion in water within a few tens of seconds and even at low average UV powers. This aluminum instability in water environments is a critical limitation. We show that the aluminum photocorrosion is related to the nonlinear absorption by water in the UV range leading to the production of hydroxyl radicals. Different corrosion protection approaches are tested using scavengers for reactive oxygen species and polymer layers deposited on top of the aluminum structures. Using optimized protection, we achieve a 10-fold increase in the available UV power range leading to no visible photocorrosion effects. This technique is crucial to achieve stable use of aluminum nanostructures enabling UV plasmonics in aqueous solutions.