1,720,952 research outputs found
Image based aberration retrieval using helical point spread functions
No full textA practical method for determining wavefront aberrations in optical systems based on the acquisition of an extended, unknown object is presented. The approach utilizes a conventional phase diversity approach in combination with a pupil-engineered, helical point spread function (PSF) to discriminate the aberrated PSF from the object features. The analysis of the image’s power cepstrum enables an efficient retrieval of the aberration coefficients by solving a simple linear system of equations. An extensive Monte Carlo simulation is performed to demonstrate that the approach makes it possible to measure low-order Zernike modes including defocus, primary astigmatism, coma, and trefoil. The presented approach is tested experimentally by retrieving the two-dimensional aberration distribution of a test setup by imaging an extended, unknown scene.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.ImPhys/Computational ImagingImPhys/Imaging Physic
Computational imaging modalities for multi-focal whole-slide imaging systems
No full textWhole-slide imaging systems can generate full-color image data of tissue slides efficiently, which are needed for digital pathology applications. This paper focuses on a scanner architecture that is based on a multi-line image sensor that is tilted with respect to the optical axis, such that every line of the sensor scans the tissue slide at a different focus level. This scanner platform is designed for imaging with continuous autofocus and inherent color registration at a throughput of the order of 400 MPx/s. Here, single-scan multi-focal whole-slide imaging, enabled by this platform, is explored. In particular, two computational imaging modalities based on multi-focal image data are studied. First, 3D imaging of thick absorption stained slides (∼60 μm) is demonstrated in combination with deconvolution to ameliorate the inherently weak contrast in thick-Tissue imaging. Second, quantitative phase tomography is demonstrated on unstained tissue slides and on fluorescently stained slides, revealing morphological features com-plementary to features made visible with conventional absorption or fluorescence stains. For both computational approaches simplified algorithms are proposed, targeted for straightforward parallel processing implementation at ∼GPx=s throughputs. Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.ImPhys/Computational ImagingImPhys/Imaging Physic
Optical trapping at low numerical aperture
Full text linkA theory of optical trapping at low Numerical Aperture (NA) is presented. The theory offers an analytical description of the competition between the stabilizing gradient and destabilizing scattering force. The trade-off can be characterized by a single dimensionless trapping parameter, which increases with bead size to wavelength ratio ?/?, m, NA and refractive index contrast m and decreases with NA. The gradient force dominates for small trapping parameters, the scattering force for large trapping parameters. The potential well depth, maximum forces and trap stiffness as a function of the three parameters (?/?, m, NA ) can be mapped onto universal functions of the trapping parameter. These functions do not depend on any free parameter. The universal well depth and maximum force curves match with numerical results based on the exact multipole expansion of the optical trapping force. The paraxial limit of low NA is relevant for compact optical tweezers based on Optical Pickup Units known from optical data storage.Imaging Science & TechnologyApplied Science
Single emitter localization analysis in the presence of background
No full textLocalization microscopy for imaging at the nano-scale relies on the quality of fitting the emitter positions from the measured light spots. The type and magnitude of the noise in the detection process, the background light level and the Point Spread Function model that is used in the fit are of paramount importance for the precision and accuracy of the fit. We present several developments on the computational methods and performance limits of single emitter localization, targeting specifically these three aspects.ImPhys/Imaging PhysicsApplied Science
Confocal Multi-line Scanning Microscope for Efficient 3D Fluorescence Imaging
No full textConfocal fluorescent imaging is the de facto standard modality for fluorescence imaging. However, the point-to-point scanning technique leads to a very limited throughput and makes the technique unsuitable for large area and fast multi-focal scanning. We propose an architecture for highly efficient 3D line confocal fluorescence imaging. Our design extends the concept of a line scanning system with continuous ‘push broom’ scanning. Instead of using a line sensor, we use an area sensor that is tilted with respect to the optical axis to acquire image data of multiple depths simultaneously. A multi-line illumination with lines illuminating the specimen at different depths, conjugate to the tilted area sensor, is created by means of a diffractive optical element (DOE). The proposed method is suitable for fast 3D image acquisition with unlimited field of view, it requires no moving components except for the sample scanning stage, has intrinsically low losses, and provides intrinsic alignment of the simultaneously scanned layers of the focal stack.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.ImPhys/Quantitative ImagingImPhys/Imaging Physic
Position and orientation estimation of fixed dipole emitters using an effective Hermite point spread function model
Full text linkWe introduce a method for determining the position and orientation of fixed dipole emitters based on a combination of polarimetry and spot shape detection. A key element is an effective Point Spread Function model based on Hermite functions. The model offers a good description of the shape variations with dipole orientation and polarization detection channel, and provides computational advantages over the exact vectorial description of dipole image formation. The realized localization uncertainty is comparable to the free dipole case in which spots are rotationally symmetric and can be well modeled with a Gaussian. This result holds for all dipole orientations, for all practical signal levels, and for defocus values within the depth of focus, implying that the massive localization bias for defocused emitters with tilted dipole axis found with Gaussian spot fitting is eliminated.IST/Imaging Science and TechnologyApplied Science
Analyzing single molecule emission patterns using Deep Learning
No full textThe time taken to generate a super-resolution image and the quality of the final synthetic image depends on the performance of the localization algorithm which is used in the localization microscopy pipeline. The most precise and accurate algorithms are mostly iterative and they take a long time to generate the localization list while the faster ‘one-shot algorithms’ are not very accurate and precise. A deep learning method smNet (single-molecule Net) was developed by Zhang et al which was claimed to perform one-shot localization with precision close to the theoretical limit and very accurately, along with performing aberration estimation and dipole-emitter orientation angle estimation. The deeplearning model smNet was trained either by augmenting experimental data or using simulated data generated with an erroneously simplified simulation model and a phase retrieval method. The purpose of this work was to characterize the performance of smNet when it was trained with simulated images generated using an accurate vector model for a range of physical conditions. Along with the characterization of smNet’s performance in doing 3D localization and aberration estimation with the accurate vector model, a pipeline was also designed which made the training process of smNet more efficient and computationally cheaper while performing accurate and precise 3D localization and aberration estimation.The pipeline was designed to implement the concept of simulator learning where a smNet model could be trained on simulated data and used to perform 3D localization and aberration estimation directly on experimental data without any retraining or domain adaptation techniques.Biomedical Engineering | Medical Physic
Data Fusion at the Nanoscale: Imaging at Resolutions Better than Wavelength/100
No full textStandard fluorescent light microscopy is rapidly approaching resolutions of a few nanometers when computationally combining information from hundreds of identical structures. We have developed algorithms to combine this information taking into account the specifics of fluorescent imaging.ImPhys/Quantitative Imagin
Particle fusion in localization microscopy
No full textSingle molecule localization microscopy (SMLM) shows promise for quantitative structural analysis of subcellular complexes and organelles with a resolution well below the diffraction limit. This superresolution microscopy technique relies on the blinking events of fluorescent molecules that labeled the structure of interest and are spatiotemporally spread over the entire field of view and time. Once hundred thousands frames of these sparse events are recorded, single molecule positions are localized with nanometer precision to form a 2D/3D point set of coordinates. Therefore, SMLM images are not conventional pixelated images but rather spatial point patterns. Photon scarcity and incomplete labeling of the imaged structure, however, limit the resolution that can possibly be achieved by means of SMLM. Moreover, due to experimental limitations the axial resolution is typically ~2-3 times worse than the lateral resolution in conventional setups. Inspired by single particle analysis (SPA) in cryo-electron microscopy (cryo-EM), proper alignment of repeated structures ("particle fusion") in a 2D/3D SMLM measurement can overcome these limiting factors and so push for isotropic resolution. The existing approaches for particle fusion in SMLM can be classified into customized routines that are borrowed from SPA in EM or methods that use strong prior knowledge about the structure to be reconstructed. While the first approaches are completely ignoring the differences in image formation model between EM and SMLM, the second ones are highly prone to the template-bias problem. In this thesis, a dedicated particle fusion pipeline for 2D/3D SMLM data is proposed. The approach properly considers the pointillistic nature of the SMLM modality and takes into account the localization uncertainties. Furthermore, while it does not require any prior knowledge about the underlying structure of the particles, it can incorporate certain features such as symmetry into the fusion process. Owing to the novel all-to-all registration scheme, the application of the devised pipeline on experimental data with very poor labeling density has been successfully demonstrated. The requirements for successful particle fusion for different SMLM modalities, namely PAINT and STORM, have been characterized through extensive study on 2D and 3D experimental and simulation data. In 2D, an FRC resolution of 3.3 nm on DNA-origami nanostructures has been achieved, and, in 3D, it was demonstrated how the combination of SMLM as a light microscopy technique and a computational approach enables structural analysis of the Nuclear Pore Complex. Future advances of SMLM rely highly on computational routines after data acquisition. Advanced data analysis techniques such as particle fusion can help pushing the boundaries of structural biology using light microscopy.ImPhys/Computational Imagin
Towards labonachip optical trapping and Raman spectroscopy of extracellular vesicles using multiwaveguide traps
No full textOptofluidic lab-on-a-chips (LOCs) employing a dual-waveguide trap for optical trapping and Raman spectroscopy have proven to be attractive and potent tools for high throughput chemical fingerprinting of bio-particles for disease diagnosis. Among the relevant bio-particles are extracellular vesicles (EVs) which a been proven through recent studies to be potential biomarkers for identification of diseases, such as cancer. However, EVs are small with diameters ranging between 30 and 1000 nm and present a challenge for both on-chip optical trapping and Raman Spectroscopy. The research presented in this thesis is aimed at the development of a multi-waveguide optical trap aimed at the combined on-chip optical trapping and Raman spectroscopy for biochemical characterisation of single EVs.Firstly, the capabilities and limitations of a dual-waveguide trap for stable on-chip optical trapping of EVs is investigated through an in-depth simulation study. This ultimately yields a comprehensive overview of stable trapping conditions for EVs in terms of EV diameter and refractive index, and the injected optical power. Then, novel multi-waveguide traps are designed and fabricated. These multi-waveguide traps lead to stronger light confinement in the channel, resulting in improved optical trapping and Raman signal generation. This is experimentally demonstrated through the optical trap stiffness values and the recorded Raman signal strength of polystyrene beads generated between a 2-waveguide and 16-waveguide trap. Finally, the 16-waveguide trap is used to demonstrate optical trapping of B. Subtillis spores, as an intermediate step towards EVs. Optical trapping of the spores is studied with both experiments and simulations. Special attention is paid to the effect of random phase differences between the beams exiting the waveguides on the optical trap quality.In conclusion, the results show promising prospects for the realisation of multi-waveguide traps for on-chip biochemical fingerprinting of EVs with optical trapping and Raman spectroscopy. <br/
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