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Journal of Visualized Experiments : JoVE Aug 2018In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the...
In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.
Topics: Antigens, Bacterial; Bacterial Toxins; Biosensing Techniques; Interferometry; Mass Spectrometry; Microscopy, Electron
PubMed: 30124667
DOI: 10.3791/57902 -
Optics Express Jun 2014There is an inherent trade-off between transverse resolution and depth of field (DOF) in optical coherence tomography (OCT) which becomes a limiting factor for certain...
There is an inherent trade-off between transverse resolution and depth of field (DOF) in optical coherence tomography (OCT) which becomes a limiting factor for certain applications. Multifocal OCT and interferometric synthetic aperture microscopy (ISAM) each provide a distinct solution to the trade-off through modification to the experiment or via post-processing, respectively. In this paper, we have solved the inverse problem of multifocal OCT and present a general algorithm for combining multiple ISAM datasets. Multifocal ISAM (MISAM) uses a regularized combination of the resampled datasets to bring advantages of both multifocal OCT and ISAM to achieve optimal transverse resolution, extended effective DOF and improved signal-to-noise ratio. We present theory, simulation and experimental results.
Topics: Algorithms; Image Enhancement; Image Interpretation, Computer-Assisted; Interferometry; Microscopy, Interference; Tomography, Optical Coherence
PubMed: 24977909
DOI: 10.1364/OE.22.016606 -
Sensors (Basel, Switzerland) Aug 2014Surface plasmon resonance (SPR) is a novel optical sensing technique with a unique ability to monitor molecular binding in real-time for biological and chemical sensor... (Review)
Review
Surface plasmon resonance (SPR) is a novel optical sensing technique with a unique ability to monitor molecular binding in real-time for biological and chemical sensor applications. Interferometry is an excellent tool for accurate measurement of SPR changes, the measurement and comparison is made for the sensitivity, dynamic range and resolution of the different analytes using interferometry techniques. SPR interferometry can also employ phase detection in addition to the amplitude of the reflected light wave, and the phase changes more rapidly compared with other approaches, i.e., intensity, angle and wavelength. Therefore, the SPR phase interferometer offers the advantages of spatial phase resolution and high sensitivity. This work discusses the advancements in interferometric SPR methods to measure the phase shifts due to refractive index changes. The main application areas of SPR sensors are demonstrated, i.e., the Fabry-Perot interferometer, Michelson interferometer and Mach-Zehnder interferometer, with different configurations. The three interferometers are discussed in detail, and solutions are suggested to enhance the performance parameters that will aid in future biological and chemical sensors.
Topics: Biosensing Techniques; Chemistry Techniques, Analytical; Equipment Design; Equipment Failure Analysis; Interferometry; Refractometry; Surface Plasmon Resonance
PubMed: 25171117
DOI: 10.3390/s140915914 -
Optics Express Sep 2022Talbot-Lau X-ray phase contrast imaging is a promising technique in biological imaging since it can provide absorption, differential phase contrast, and dark-field...
Talbot-Lau X-ray phase contrast imaging is a promising technique in biological imaging since it can provide absorption, differential phase contrast, and dark-field images simultaneously. However, high accuracy motorized translation stages and high stability of the imaging system are needed to avoid moiré artifacts in the reconstructed images. In this work, the effects of the stepping errors and the dose fluctuations on the transmission, differential phase contrast, and dark-field images are theoretically derived and systematically summarized. A novel three-step iterative method is designed for image reconstruction in Talbot-Lau interferometry with phase-stepping errors and dose fluctuations. Phase distributions, phase-stepping errors, and dose fluctuation coefficients are iteratively updated via the least square method until the convergence criteria are met. Moiré artifacts are mostly reduced via the proposed method in both the numerical simulations and experiments. The reconstructed images are highly coincident with the ground truth, which verifies the high accuracy of this method. The proposed algorithm is also compared with other moiré artifacts reduction algorithms, which further demonstrates the high precision of this algorithm. This work is beneficial for reducing the strict requirements for the hardware system in the conventional Talbot-Lau interferometry, such as the high accuracy motorized stages and the X-ray tube with high stability, which is significant for advancing the X-ray phase contrast imaging towards the practical medical applications.
Topics: Artifacts; X-Rays; Radiography; Interferometry; Image Processing, Computer-Assisted
PubMed: 36258469
DOI: 10.1364/OE.466277 -
Optics Express Aug 2022In this manuscript, we describe the development of a single shot, self-referencing wavefront division, multiplexing digital holographic microscope employing LED sources...
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.
Topics: Humans; Microscopy, Phase-Contrast; Polystyrenes; Holography; Interferometry; Erythrocytes
PubMed: 36299102
DOI: 10.1364/OE.444616 -
Optics Express Dec 2022Ultrasound-modulated optical tomography (UOT), which combines the advantages of both light and ultrasound, is a promising imaging modality for deep-tissue...
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.
Topics: Lighting; Phantoms, Imaging; Ultrasonography; Tomography, Optical; Interferometry
PubMed: 36558581
DOI: 10.1364/OE.470956 -
Medical Physics Nov 2015Grating interferometry is a state-of-the-art x-ray imaging approach, which can acquire information on x-ray attenuation, phase shift, and small-angle scattering...
PURPOSE
Grating interferometry is a state-of-the-art x-ray imaging approach, which can acquire information on x-ray attenuation, phase shift, and small-angle scattering simultaneously. Phase-contrast imaging and dark-field imaging are very sensitive to microstructural variation and offers superior contrast resolution for biological soft tissues. However, a common x-ray tube is a point-like source. As a result, the popular planar grating imaging configuration seriously restricts the flux of photons and decreases the visibility of signals, yielding a limited field of view. The purpose of this study is to extend the planar x-ray grating imaging theory and methods to a spherical grating scheme for a wider range of preclinical and clinical applications.
METHODS
A spherical grating matches the wave front of a point x-ray source very well, allowing the perpendicular incidence of x-rays on the grating to achieve a higher visibility over a larger field of view than the planer grating counterpart. A theoretical analysis of the Talbot effect for spherical grating imaging is proposed to establish a basic foundation for x-ray spherical gratings interferometry. An efficient method of spherical grating imaging is also presented to extract attenuation, differential phase, and dark-field images in the x-ray spherical grating interferometer.
RESULTS
Talbot self-imaging with spherical gratings is analyzed based on the Rayleigh-Sommerfeld diffraction formula, featuring a periodic angular distribution in a polar coordinate system. The Talbot distance is derived to reveal the Talbot self-imaging pattern. Numerical simulation results show the self-imaging phenomenon of a spherical grating interferometer, which is in agreement with the theoretical prediction.
CONCLUSIONS
X-ray Talbot interferometry with spherical gratings has a significant practical promise. Relative to planar grating imaging, spherical grating based x-ray Talbot interferometry has a larger field of view and improves both signal visibility and dose utilization for pre-clinical and clinical applications.
Topics: Computer Simulation; Computer-Aided Design; Equipment Design; Equipment Failure Analysis; Interferometry; Models, Theoretical; Refractometry; Scattering, Radiation; X-Ray Diffraction; X-Rays
PubMed: 26520741
DOI: 10.1118/1.4933195 -
Analytical and Bioanalytical Chemistry May 2020Direct optical detection has proven to be a highly interesting tool in biomolecular interaction analysis to be used in drug discovery, ligand/receptor interactions,... (Review)
Review
Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects.
Direct optical detection has proven to be a highly interesting tool in biomolecular interaction analysis to be used in drug discovery, ligand/receptor interactions, environmental analysis, clinical diagnostics, screening of large data volumes in immunology, cancer therapy, or personalized medicine. In this review, the fundamental optical principles and applications are reviewed. Devices are based on concepts such as refractometry, evanescent field, waveguides modes, reflectometry, resonance and/or interference. They are realized in ring resonators; prism couplers; surface plasmon resonance; resonant mirror; Bragg grating; grating couplers; photonic crystals, Mach-Zehnder, Young, Hartman interferometers; backscattering; ellipsometry; or reflectance interferometry. The physical theories of various optical principles have already been reviewed in detail elsewhere and are therefore only cited. This review provides an overall survey on the application of these methods in direct optical biosensing. The "historical" development of the main principles is given to understand the various, and sometimes only slightly modified variations published as "new" methods or the use of a new acronym and commercialization by different companies. Improvement of optics is only one way to increase the quality of biosensors. Additional essential aspects are the surface modification of transducers, immobilization strategies, selection of recognition elements, the influence of non-specific interaction, selectivity, and sensitivity. Furthermore, papers use for reporting minimal amounts of detectable analyte terms such as value of mass, moles, grams, or mol/L which are difficult to compare. Both these essential aspects (i.e., biochemistry and the presentation of LOD values) can be discussed only in brief (but references are provided) in order to prevent the paper from becoming too long. The review will concentrate on a comparison of the optical methods, their application, and the resulting bioanalytical quality.
Topics: Animals; Biosensing Techniques; Equipment Design; Humans; Interferometry; Light; Optical Devices; Refractometry; Surface Plasmon Resonance; Transducers
PubMed: 32313998
DOI: 10.1007/s00216-020-02581-0 -
Optics Express May 2008Common-path in-line shearing interferometry, combined with pixel-array imaging, provides a surface metrology that achieves 15 pm surface height resolution. An...
Common-path in-line shearing interferometry, combined with pixel-array imaging, provides a surface metrology that achieves 15 pm surface height resolution. An eighth-wave thermal oxide on silicon generates a reference wave locked in the condition of phase quadrature for phase-to-intensity conversion that makes surface height or index variations directly detectable by an imaging system. The scaling surface mass sensitivity for the surface metrology application is S(scal) = 7 fg/mm under 40x magnification with a molecular resolution of approximately 12 IgG molecules within a pixel, limited by the surface roughness of the substrate. When applied to reverse-phase immunoassays in an antibody microarray format under 7x magnification, the current limit of detection is 10 ng/ml for 1 hour incubation, limited by biological and chemical variability. The biosensor is compatible with real-time binding measurements under active flow conditions with a binding dynamic range per well of 10(3) and a mass sensitivity of 2 pg/mm(2).
Topics: Animals; Antibodies; Biosensing Techniques; Equipment Design; Humans; Immunoassay; Immunoglobulin G; Interferometry; Light; Mice; Optics and Photonics; Oxides; Proteome; Rabbits; Reproducibility of Results
PubMed: 18545414
DOI: 10.1364/oe.16.007102 -
Optics Express Jan 2008The stability of the Shack-Hartmann sensor against mechanical disturbances from the environment can advantageously be exploited in highly sensitive wave front tests of...
The stability of the Shack-Hartmann sensor against mechanical disturbances from the environment can advantageously be exploited in highly sensitive wave front tests of surfaces. Here, a Fizeau-type multiple beam test is investigated. The enhancement of the phase sensitivity in a Fizeau-resonator formed by two high reflecting mirror surfaces enables tests in reflected light which includes also opaque surfaces. The multiplication of the sensitivity with the number of passes through the Fizeau resonator provides a big margin against the rather limited repeatability of wave front measurements with such a wave front sensor. The method has been tested for planeness Fizeau measurements. It could also be exploited in spherical Fizeau tests. But in the latter case the two spherical surfaces forming the resonator should have radii which differ only by e.g. 1mm.
Topics: Equipment Design; Equipment Failure Analysis; Interferometry; Refractometry
PubMed: 18521168
DOI: 10.1364/oe.16.000362