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.
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.
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.
The fundamental understanding of molecular quantum electrodynamics via the strong light–matter interactions between a nanophotonic cavity and quantum emitters opens various applications in quantum ...biology, biophysics, and chemistry. However, considerable obstacles to obtaining a clear understanding of coupling mechanisms via reliable experimental quantifications remain to be resolved before this field can truly blossom toward practical applications in quantitative life science and photochemistry. Here, we provide recent advancements of state-of-the-art demonstrations in plexcitonic and vibro-polaritonic strong couplings and their applications. We highlight recent studies on various strong coupling systems for altering chemical reaction landscapes. Then, we discuss reports dedicated to the utilization of strong coupling methods for biomolecular sensing, protein functioning studies, and the generation of hybrid light–matter states inside living cells. The strong coupling regime provides a tool for investigating and altering coherent quantum processes in natural biological processes. We also provide an overview of new findings and future avenues of quantum biology and biochemistry.
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.
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.
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.
Multiplexed metasurface–driven plasmonic resonance energy transfer (PRET) hyperspectral imaging is created to probe biological light–matter interactions. Pixelated metasurfaces with engineered scattering spectra over the entire visible range, by the precision nanoengineering of gap plasmon and optical effects of metasurface clusters, are designed, fabricated, characterized by their full dark‐field coloration, and are demonstrated for multiplexed real‐time molecular sensing and imaging methods.
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 β-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.
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.