RESUMO
Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2 nm axial localization precision, well below the few-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E. coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.
Assuntos
Interferometria/métodos , Imagem Individual de Molécula/métodos , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Humanos , Imageamento Tridimensional/métodosRESUMO
Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rß1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.
Assuntos
Subunidade beta 1 de Receptor de Interleucina-12/metabolismo , Interleucina-23/metabolismo , Receptores de Interleucina/metabolismo , Animais , Calorimetria/métodos , Linhagem Celular , Humanos , Interferometria/métodos , Subunidade p40 da Interleucina-12/metabolismo , Masculino , Camundongos , Ligação Proteica/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
X-ray computed tomography (CT) is one of the most commonly used three-dimensional medical imaging modalities today. It has been refined over several decades, with the most recent innovations including dual-energy and spectral photon-counting technologies. Nevertheless, it has been discovered that wave-optical contrast mechanisms-beyond the presently used X-ray attenuation-offer the potential of complementary information, particularly on otherwise unresolved tissue microstructure. One such approach is dark-field imaging, which has recently been introduced and already demonstrated significantly improved radiological benefit in small-animal models, especially for lung diseases. Until now, however, dark-field CT could not yet be translated to the human scale and has been restricted to benchtop and small-animal systems, with scan durations of several minutes or more. This is mainly because the adaption and upscaling to the mechanical complexity, speed, and size of a human CT scanner so far remained an unsolved challenge. Here, we now report the successful integration of a Talbot-Lau interferometer into a clinical CT gantry and present dark-field CT results of a human-sized anthropomorphic body phantom, reconstructed from a single rotation scan performed in 1 s. Moreover, we present our key hardware and software solutions to the previously unsolved roadblocks, which so far have kept dark-field CT from being translated from the optical bench into a rapidly rotating CT gantry, with all its associated challenges like vibrations, continuous rotation, and large field of view. This development enables clinical dark-field CT studies with human patients in the near future.
Assuntos
Espalhamento a Baixo Ângulo , Tomografia Computadorizada por Raios X/instrumentação , Tomografia Computadorizada por Raios X/métodos , Algoritmos , Animais , Humanos , Imageamento Tridimensional , Interferometria/métodos , Imagens de Fantasmas , Radiografia , Tomógrafos Computadorizados , Raios XRESUMO
Bladder cancer is one of the most common cancers with a high recurrence rate. Patients undergo mandatory yearly scrutinies, including cystoscopies, which makes bladder cancer highly distressing and costly. Here, we aim to develop a non-invasive, label-free method for the detection of bladder cancer cells in urine samples, which is based on interferometric imaging flow cytometry. Eight urothelial carcinoma and one normal urothelial cell lines, along with red and white blood cells, imaged quantitatively without staining by an interferometric phase microscopy module while flowing in a microfluidic chip, and classified by two machine-learning algorithms, based on deep-learning semantic segmentation convolutional neural network and extreme gradient boosting. Furthermore, urine samples obtained from bladder-cancer patients and healthy volunteers were imaged, and classified by the system. We achieved accuracy and area under the curve (AUC) of 99% and 97% for the cell lines on both machine-learning algorithms. For the real urine samples, the accuracy and AUC were 96% and 96% for the deep-learning algorithm and 95% and 93% for the gradient-boosting algorithm, respectively. By combining label-free interferometric imaging flow cytometry with high-end classification algorithms, we achieved high-performance differentiation between healthy and malignant cells. The proposed technique has the potential to supplant cystoscopy in the bladder cancer surveillance and diagnosis space.
Assuntos
Citometria de Fluxo , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/urina , Citometria de Fluxo/métodos , Linhagem Celular Tumoral , Interferometria/métodos , Algoritmos , Aprendizado de Máquina , Aprendizado ProfundoRESUMO
The preservation method to store bone tissue for posterior analysis is a widespread practice. However, the method's potential influence on the material's mechanical properties is often overlooked during single-point experimentation. Saline and formaldehyde solutions are the most common among the employed preservation media. A full field analysis of the mice femoral bone deformation using non-destructive optical techniques is conducted to assess the influence of the storage media on the viscoelastic properties of the tissue. Three different groups are subjected to a standard three-point bending test. The first group is the control, with fresh post-mortem samples. The second and third groups used saline and formaldehyde solutions, respectively. During the mechanical test, the bone's surface and internal deformation are monitored simultaneously using digital holographic interferometry and Fourier-domain optical coherence tomography. A mechanical comparison among the three groups is presented. The results show that after 48 h of immersion in saline solution, the mice bones keep their viscoelastic behavior similar to fresh bones. Meanwhile, 48 h in formaldehyde modifies the response and affects the marrow structure. The high sensitivity of the optical phase also makes it possible to observe changes in the anisotropy of the samples. As a comparison, Raman spectroscopy analyzes the three bone groups to prove that the preservation media does not affect a single-point inspection.
Assuntos
Fêmur , Formaldeído , Análise Espectral Raman , Tomografia de Coerência Óptica , Animais , Camundongos , Formaldeído/farmacologia , Tomografia de Coerência Óptica/métodos , Análise Espectral Raman/métodos , Fêmur/diagnóstico por imagem , Fêmur/fisiologia , Testes Mecânicos , Elasticidade/efeitos dos fármacos , Viscosidade , Soluções para Preservação de Órgãos/farmacologia , Interferometria/métodos , Solução SalinaRESUMO
PURPOSE: To determine whether visible light is needed to elicit axial eye shortening by exposure to long wavelength light. METHODS: Incoherent narrow-band red (620 ± 10 nm) or near-infrared (NIR, 875 ± 30 nm) light was generated by an array of light-emitting diodes (LEDs) and projected monocularly in 17 myopic and 13 non-myopic subjects for 10 min. The fellow eye was occluded. Light sources were positioned 50 cm from the eye in a dark room. Axial length (AL) was measured before and after the exposure using low-coherence interferometry. RESULTS: Non-myopic subjects responded to red light with significant eye shortening, while NIR light induced minor axial elongation (-13.3 ± 17.3 µm vs. +6.5 ± 11.6 µm, respectively, p = 0.005). Only 41% of the myopic subjects responded to red light exposure with a decrease in AL and changes were therefore, on average, not significantly different from those observed with NIR light (+0.2 ± 12.1 µm vs. +1.1 ± 11.2 µm, respectively, p = 0.83). Interestingly, there was a significant correlation between refractive error and induced changes in AL after exposure to NIR light in myopic eyes (r(15) = -0.52, p = 0.03) and induced changes in AL after exposure to red light in non-myopic eyes (r(11) = 0.62, p = 0.02), with more induced axial elongation with increasing refractive error. CONCLUSIONS: Incoherent narrow-band red light at 620 nm induced axial shortening in 77% of non-myopic and 41% of myopic eyes. NIR light did not induce any significant changes in AL in either refractive group, suggesting that the beneficial effect of red laser light therapy on myopia progression requires visible stimulation and not simply thermal energy.
Assuntos
Comprimento Axial do Olho , Raios Infravermelhos , Miopia , Humanos , Comprimento Axial do Olho/diagnóstico por imagem , Miopia/fisiopatologia , Masculino , Feminino , Raios Infravermelhos/efeitos adversos , Adulto , Adulto Jovem , Interferometria/métodos , Refração Ocular/fisiologia , Luz/efeitos adversos , AdolescenteRESUMO
The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.
Assuntos
Proteínas , Ressonância de Plasmônio de Superfície , Ligação Proteica , Termodinâmica , Proteínas/química , Interferometria/métodos , CinéticaRESUMO
Numerical simulations of head-related transfer functions (HRTFs) conventionally assume a rigid boundary condition for the pinna. The human pinna, however, is an elastic deformable body that can vibrate due to incident acoustic waves. This work investigates how sound-induced vibrations of the pinna can affect simulated HRTF magnitudes. The work will motivate the research question by measuring the sound-induced vibrational patterns of an artificial pinna with a high-speed holographic interferometric system. Then, finite element simulations are used to determine HRTFs for a tabletop model of the B&K 5128 head and torso simulator for a number of directions. Two scenarios are explored: one where the pinna is modeled as perfectly rigid, and another where the pinna is modeled as linear elastic with material properties close to that of auricular cartilage. The findings suggest that pinna vibrations have negligible effects on HRTF magnitudes up to 5 kHz. The same conclusion, albeit with less certainty, is drawn for higher frequencies. Finally, the importance of the elastic domain's material properties is emphasized and possible implications for validation studies on dummy heads 1as well as the limitations of the present work are discussed in detail.
Assuntos
Simulação por Computador , Pavilhão Auricular , Análise de Elementos Finitos , Cabeça , Som , Vibração , Humanos , Pavilhão Auricular/fisiologia , Pavilhão Auricular/anatomia & histologia , Cabeça/fisiologia , Cabeça/anatomia & histologia , Holografia/métodos , Interferometria/métodos , Elasticidade , Análise Numérica Assistida por Computador , Modelos Biológicos , Movimento (Física) , Estimulação AcústicaRESUMO
The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/química , Glicoproteína da Espícula de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/efeitos dos fármacos , Humanos , COVID-19/virologia , COVID-19/metabolismo , Interferometria/métodos , Citometria de Fluxo/métodosRESUMO
Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.
Assuntos
Lectinas , Ramnose , Lectinas/química , Carboidratos/química , Proteínas Recombinantes/genética , Interferometria/métodosRESUMO
Spatial coherence of light sources is usually obtained by using the classical Young's interferometer. Although the original experiment was improved upon in successive works, some drawbacks still remain. For example, several pairs of points must be used to obtain the complex coherence degree (normalized first-order correlation function) of the source. In this work, a modified Mach-Zehnder interferometer which includes a pair of lenses and is able to measure the spatial coherence degree is presented. With this modified Mach-Zehnder interferometer, it is possible to measure the full 4D spatial coherence function by displacing the incoming beam laterally. To test it, we have measured only a 2D projection (zero shear) of the 4D spatial coherence, which is enough to characterize some types of sources. The setup has no movable parts, making it robust and portable. To test it, the two-dimensional spatial coherence of a high-speed laser with two cavities was measured for different pulse energy values. We observe from the experimental measurements that the complex degree of coherence changes with the selected output energy. Both laser cavities seem to have similar complex coherence degrees for the maximum energy, although it is not symmetrical. Thus, this analysis will allow us to determine the best configuration of the double-cavity laser for interferometric applications. Furthermore, the proposed approach can be applied to any other light sources.
Assuntos
Lasers , Lentes , Interferometria/métodosRESUMO
Interferometry is a prime technique for modern precision measurements. Atoms, unlike light, have significant interactions with electric, magnetic, and gravitational fields, making their use in interferometric applications particularly versatile. Here, we demonstrate atom interferometry to image optical and magnetic potential landscapes over an area exceeding 240 µm×600 µm. The differential potentials employed in our experiments generate phase imprints in an atom laser that are made visible through a Ramsey pulse sequence. We further demonstrate how advanced pulse sequences can enhance desired imaging features, e.g., to image steep potential gradients. A theoretical discussion is presented that provides a semiclassical analysis and matching numerics.
Assuntos
Interferometria , Lasers , Interferometria/métodos , LuzRESUMO
Neurons undergo nanometer-scale deformations during action potentials, and the underlying mechanism has been actively debated for decades. Previous observations were limited to a single spot or the cell boundary, while movement across the entire neuron during the action potential remained unclear. Here we report full-field imaging of cellular deformations accompanying the action potential in mammalian neuron somas (-1.8 to 1.4 nm) and neurites (-0.7 to 0.9 nm), using high-speed quantitative phase imaging with a temporal resolution of 0.1 ms and an optical path length sensitivity of <4 pm per pixel. The spike-triggered average, synchronized to electrical recording, demonstrates that the time course of the optical phase changes closely matches the dynamics of the electrical signal. Utilizing the spatial and temporal correlations of the phase signals across the cell, we enhance the detection and segmentation of spiking cells compared to the shot-noise-limited performance of single pixels. Using three-dimensional (3D) cellular morphology extracted via confocal microscopy, we demonstrate that the voltage-dependent changes in the membrane tension induced by ionic repulsion can explain the magnitude, time course, and spatial features of the phase imaging. Our full-field observations of the spike-induced deformations shed light upon the electromechanical coupling mechanism in electrogenic cells and open the door to noninvasive label-free imaging of neural signaling.
Assuntos
Potenciais de Ação , Membrana Celular/fisiologia , Interferometria/métodos , Neurônios/citologia , Neurônios/fisiologia , Animais , Imagem Molecular , OptogenéticaRESUMO
Biolayer interferometry (BLI) is a technology which allows to study the affinity between two interacting macro-molecules and to visualize their kinetic of interaction in real time. In this work, we combine BLI interaction measurement with mass spectrometry in order to identify the proteins interacting with the bait. We provide for the first time the proof of concept of the feasibility of BLI-MS in complex biological mixtures.
Assuntos
Interferometria , Proteínas , Interferometria/métodos , Cinética , Espectrometria de Massas , Proteínas/químicaRESUMO
The study of multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Biolayer interferometry is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous isothermal titration calorimetry experiments, and presented strong advantages because they require less quantity and purity for the biomolecules.
Assuntos
Glicoconjugados , Lectinas , Dendrímeros/química , Glicoconjugados/química , Interferometria/métodos , Lectinas/química , LigantesRESUMO
Ultrasound-modulated optical tomography (UOT), which combines the advantages of both light and ultrasound, is a promising imaging modality for deep-tissue high-resolution imaging. Among existing implementations, camera-based UOT gains huge advances in modulation depth through parallel detection. However, limited by the long exposure time and the slow framerate of modern cameras, the measurement of UOT signals always requires holographic methods with additional reference beams. This requirement increases system complexity and is susceptible to environmental disturbances. To overcome this challenge, we develop coaxial interferometry for camera-based UOT in this work. Such a coaxial scheme is enabled by employing paired illumination with slightly different optical frequencies. To measure the UOT signal, the conventional phase-stepping method in holography can be directly transplanted into coaxial interferometry. Specifically, we performed both numerical investigations and experimental validations for camera-based UOT under the proposed coaxial scheme. One-dimensional imaging for an absorptive target buried inside a scattering medium was demonstrated. With coaxial interferometry, this work presents an effective way to reduce system complexity and cope with environmental disturbances for camera-based UOT.
Assuntos
Iluminação , Tomografia Óptica , Imagens de Fantasmas , Ultrassonografia/métodos , Tomografia Óptica/métodos , Interferometria/métodosRESUMO
Interferometric scattering (iSCAT) microscopy is a highly sensitive imaging technique that uses common-path interferometry to detect the linear scattering fields associated with samples. However, when measuring a complex sample, such as a biological cell, the superposition of the scattering signals from various sources, particularly those along the optical axis of the microscope objective, considerably complicates the data interpretation. Herein, we demonstrate high-speed, wide-field iSCAT microscopy in conjunction with confocal optical sectioning. Utilizing the multibeam scanning strategy of spinning disk confocal microscopy, our iSCAT confocal microscope acquires images at a rate of 1,000 frames per second (fps). The configurations of the spinning disk and the background correction procedures are described. The iSCAT confocal microscope is highly sensitive-individual 10 nm gold nanoparticles are successfully detected. Using high-speed iSCAT confocal imaging, we captured the rapid movements of single nanoparticles on the model membrane and single native vesicles in the living cells. Label-free iSCAT confocal imaging enables the detailed visualization of nanoscopic cell dynamics in their most native forms. This holds promise to unveil cell activities that are previously undescribed by fluorescence-based microscopy.
Assuntos
Ouro , Nanopartículas Metálicas , Microscopia Confocal/métodos , Interferometria/métodos , Microscopia de Fluorescência/métodosRESUMO
Most optical characterization methods rely on measuring the complex optical fields emerging from the interaction between light and material systems. Nevertheless, inherent scattering and absorption cause ambiguities in both interferometric and noninterferometric attempts to measure phase. Here we demonstrate that the complete information about a probe optical field can be encoded into the states of polarization, and develop a topography measurement method by blindly varying the ambient refractive index surrounding the sample in a wedged cuvette, which is capable of simultaneously measuring the thickness and the ambient refractive index of the sample in real time, as well as extending the measurement range of the sample thickness. With the method, we have successfully measured the topography of a 136.7 µm thick coverslip by blindly changing the ambient refractive index by 0.001246, resulting in the thickest sample characterization ever achieved by quantitative phase imaging, to the best of our knowledge. An efficient and complete characterization of optical fields is critical for any high-resolution imaging approach and the technique demonstrated here should prove attractive for applications ranging from microscopy to remote sensing. Thanks to the high precision and fast response speed, this method may pave a new way for measuring the topography of the thick samples, such as biological tissues.
Assuntos
Interferometria , Refratometria , Refratometria/métodos , Interferometria/métodos , Microscopia/métodosRESUMO
In this manuscript, we describe the development of a single shot, self-referencing wavefront division, multiplexing digital holographic microscope employing LED sources for large field of view quantitative phase imaging of biological samples. To address the difficulties arising while performing interferometry with low temporally coherent sources, an optical arrangement utilizing multiple Fresnel Biprisms is used for hologram multiplexing, enhancing the field of view and increasing the signal to noise ratio. Biprisms offers the ease of obtaining interference patterns by automatically matching the path length between the two off-axis beams. The use of low temporally coherent sources reduces the speckle noise and the cost, and the form factor of the setup. The developed technique was implemented using both visible and UV LEDs and tested on polystyrene microspheres and human erythrocytes.
Assuntos
Holografia , Poliestirenos , Humanos , Microscopia de Contraste de Fase , Holografia/métodos , Interferometria/métodos , EritrócitosRESUMO
It is always a challenge how to overcome speckle noise interference in the phase reconstruction for coherent digital holography (CDH) and its application, as this issue has not been solved well so far. In this paper, we are proposing an enhanced anti-speckle deep neural unwrapping network (E-ASDNUN) approach to achieve high quality of absolute phase reconstruction for CDH. The method designs a special network-based noise filter and embeds it into a deep neural unwrapping network to enhance anti-noise capacity in the image feature recognition and extraction process. The numerical simulation and experimental test on the phase unwrapping reconstruction and the image quality evaluation under the noise circumstances show that the E-ASDNUN approach is very effective against the speckle noise in realizing the high quality of absolute phase reconstruction. Meanwhile, it also demonstrates much better robustness than the typical U-net neural network and the traditional phase unwrapping algorithms in reconstructing high wrapping densities and high noise levels of phase images. The E-ASDNUN approach is also examined and confirmed by measuring the same phase object using a commercial white light interferometry as a reference. The result is perfectly consistent with that obtained by the E-ASDNUN approach.