Extending single-molecule microscopy using optical Fourier processing.
Ontology highlight
ABSTRACT:
SUBMITTER: Backer AS
PROVIDER: S-EPMC4317050 | biostudies-other | 2014 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:In this Letter, we propose a new quantitative phase imaging methodology named Fourier optical spin splitting microscopy (FOSSM). FOSSM relies on a metasurface located at the Fourier plane of a polarized microscope to separate the object image into two replicas of opposite circularly polarized states. The bias retardation between the two replicas is tuned by translating the metasurface or rotating the analyzer. Combined with a polarized camera, FOSSM can easily achieve single-shot quantitative phase gradient imaging, which greatly reduces the complexity of current phase microscope setups, paving the way for the next generation high-speed real-time multifunctional microscopy.
| S-EPMC10035159 | biostudies-literature
Project description:The development and use of interferometric variable-polarization Fourier transform nonlinear optical (vpFT-NLO) imaging to distinguish colloidal nanoparticles colocated within the optical diffraction limit is described. Using a collinear train of phase-stabilized pulse pairs with orthogonal electric field vectors, the polarization of nonlinear excitation fields are controllably modulated between linear, circular, and various elliptical states. Polarization modulation is achieved by precise control over the time delay separating the orthogonal pulse pairs to within hundreds of attoseconds. The resultant emission from gold nanorods is imaged to a 2D array detector and correlated to the excitation field polarization and plasmon resonance frequency by Fourier transformation. Gold nanorods with length-to-diameter aspect ratios of 2 support a longitudinal surface plasmon resonance at approximately 800 nm, which is resonant with the excitation fundamental carrier wavelength. Differences in the intrinsic linear and circular dichroism resulting from variation in their relative alignment with respect to the laboratory frame enable optical differentiation of nanorods separated within 50 nm, which is an approximate 5-fold improvement over the diffraction limit of the microscope. The experimental results are supported by analytical simulations. In addition to subdiffraction spatial resolution, the vpFT-NLO method intrinsically provides the polarization- and frequency-dependent resonance response of the nanoparticles-providing spectroscopic information content along with super-resolution imaging capabilities.
| S-EPMC10131489 | biostudies-literature
Project description:We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.
| S-EPMC5540136 | biostudies-literature
Project description:Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.
| S-EPMC3397402 | biostudies-literature
Project description:Visualizing the optical response of individual molecules is a long-standing goal in catalysis, molecular nanotechnology, and biotechnology. The molecular response is dominated not only by the electronic states in their isolated environment but also by neighboring molecules and the substrate. Information about the transfer of energy and charge in real environments is essential for the design of the desired molecular functions. However, visualizing these factors with spatial resolution beyond the molecular scale has been challenging. Here, by combining photoinduced force microscopy and Kelvin probe force microscopy, we have mapped the photoinduced force in a pentacene bilayer with a spatial resolution of 0.6 nm and observed its "multipole excitation". We identified the excitation as the result of energy and charge transfer between the molecules and to the Ag substrate. These findings can be achieved only by combining microscopy techniques to simultaneously visualize the optical response of the molecules and the charge transfer between the neighboring environments. Our approach and findings provide insights into designing molecular functions by considering the optical response at each step of layering molecules.
| S-EPMC10795473 | biostudies-literature
Project description:Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.
| S-EPMC3465359 | biostudies-other
Project description:The resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2-3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.
| S-EPMC5428856 | biostudies-literature
Project description:Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.
| S-EPMC9160414 | biostudies-literature
Project description:Single-molecule localization-based super-resolution microscopy has enabled the imaging of microscopic objects beyond the diffraction limit. However, this technique is limited by the requirements of imaging an extremely large number of frames of biological samples to generate a super-resolution image, thus requiring a longer acquisition time. Additionally, the processing of such a large image sequence leads to longer data processing time. Therefore, accelerating image acquisition and processing in single-molecule localization microscopy (SMLM) has been of perennial interest. To accelerate three-dimensional (3D) SMLM imaging by leveraging a computational approach without compromising the resolution. We used blind sparse inpainting to reconstruct high-density 3D images from low-density ones. The low-density images are generated using much fewer frames than usually needed, thus requiring a shorter acquisition and processing time. Therefore, our technique will accelerate 3D SMLM without changing the existing standard SMLM hardware system and labeling protocol. The performance of the blind sparse inpainting was evaluated on both simulation and experimental datasets. Superior reconstruction results of 3D SMLM images using up to 10-fold fewer frames in simulation and up to 50-fold fewer frames in experimental data were achieved. We demonstrate the feasibility of fast 3D SMLM imaging leveraging a computational approach to reduce the number of acquired frames. We anticipate our technique will enable future real-time live-cell 3D imaging to investigate complex nanoscopic biological structures and their functions.
| S-EPMC7910702 | biostudies-literature
Project description:Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.
| S-EPMC8080769 | biostudies-literature