Point Scanning Research Articles

Direct laser interference patterning via scanning optics using the Michelson-Morley configuration

Direct Laser Interference Patterning (DLIP) is a promising method to realize patterns on surfaces, based on two or more beams, interference. Compared with the traditional direct laser writing approach, DLIP allows control of the features’ shape and size below the size of the laser beam. The standard configuration of a DLIP system is based on fixed optical elements and the movement of the sample via a motorized axis. This work proposes a new optical setup for two-beam DLIP. A Michelson-Morley interferometer was used, realizing the interference of a split beam. A nanosecond pulsed green laser was used to realize the interference phenomenon. The innovative concept combines the interferometer with an industrial scanner head for beam steering. The work explores possibility to use the galvanometric mirrors in DLIP to expand the process capabilities towards larger areas without moving the workpiece. Moreover, the concept is adaptable for different laser wavelengths and optical solutions permitting a degree of flexibility in the pattern period. An optical model is proposed for the first time to describe the design requirements and the pattern period for scanning optics. The developed prototype was used to pattern a biodegradable magnesium alloy with known difficulties for nanosecond pulse ablation. Two strategies namely point by point and continuous scanning were investigated. Periodic linear patterns were produced with periodicity equal to 23 µm and with a productivity up to 6.5 cm2/min.

Multifocal confocal microscopy using a volume holographic lenslet array illuminator.

Multifocal illumination can improve image acquisition time compared to single point scanning in confocal microscopy. However, due to an increase in the system complexity, obtaining uniform multifocal illumination throughout the field of view with conventional methods is challenging. Here, we propose a volume holographic lenslet array illuminator (VHLAI) for multifocal confocal microscopy. To obtain uniform array illumination, a super Gaussian (SG) beam has been incorporated through VHLAI with an efficiency of 43%, and implemented in a confocal microscope. The design method for a photo-polymer based volume holographic beam shaper is presented and its advantages are thoroughly addressed. The proposed system can significantly improve image acquisition time without sacrificing the quality of the image. The performance of the proposed multifocal confocal microscopy was compared with wide-field images and also evaluated by measuring optically sectioned microscopic images of fluorescence beads, florescence pollen grains, and biological samples. The proposed multifocal confocal system generates images faster without any changes in scanning devices. The present method may find important applications in high-speed multifocal microscopy platforms.

Investigating the Performances of Wide-Field Raman Microscopy with Stochastic Optical Reconstruction Post-Processing.

Super-resolution fluorescence microscopy based on localization algorithms has tremendously impacted the field of imaging by improving the spatial resolution of optical measurements with specific blinking fluorophores and concomitant reduction of acquisition time. In vibrational spectroscopy and imaging, various methods have been developed to surpass the diffraction limit including near-field scattering methods, such as in tip-enhanced Raman and infrared spectroscopies. Although these scanning-probe techniques can provide exquisite spatial resolution, they often require long acquisition times and tedious fabrication of nano-scale scanning probes. Herein, stochastic optical reconstruction microscopy (STORM) protocol is applied on Raman measurements acquired using a wide-field home-built microscopy setup. We explore how the fluctuations of the Raman signal acquired over a series of time-lapse images at specific spectral ranges can be exploited with STORM processing, possibly revealing details with improved spatial resolution, under lower irradiance and with faster acquisition speed that cannot be achieved in point scanning mode over the same field of view. Samples studied here include patterned silicon, polystyrene microspheres on a silicon wafer, and graphene on a silicon/silicon dioxide substrate. The outcome presents an effective way to collect Raman images at selected spectral ranges with spatial resolutions of ∼200 nm over a large field of view under 532 nm excitation together with an acquisition speed improved by two orders of magnitude and under a significantly reduced irradiance compared to confocal laser scanning acquisition.

Application of Near-Field Scanning Microwave Microscopy in Liquid Environment

In this article, a method of illustrating the electromagnetic properties of liquid specimens is proposed by our homemade near-field scanning microwave microscopy (NSMM). By introducing the fundamental theorem of NSMM and conducting a simulation on the configuration of the whole system, parameters including quality factor and resonant frequency in electrolyte liquid specimen vary with types of the solution as well as their concentration. By conducting the line scanning testing method, the relation of concentration of a specified solution concerning its corresponding electromagnetic properties is clarified. Eventually, yeast cells are tested at living or dead status with point scanning and line scanning separately. The NSMM experiment results exhibit a substantial capacity for biologic samples in a liquid environment.

Challenging Point Scanning across Electron Microscopy and Optical Imaging using Computational Imaging

Solving challenges of enhanced imaging (resolution or speed) is a continuously changing frontier of research. Within this sphere, ghost imaging (and the closely related single-pixel imaging) has evolved as an alternative to focal plane detector arrays owing to advances in detectors and/or modulation devices. The interest in these techniques is due to their robustness to varied sets of patterns and applicability to a broad range of wavelengths and compatibility with compressive sensing. To achieve a better control of illumination strategies, modulators of many kinds have long been available in the optical regime. However, analogous technology to control of phase and amplitude of electron beams does not exist. We approach this electron microscopy challenge from an optics perspective, with a novel approach to imaging with non-orthogonal pattern sets using ghost imaging. Assessed first in the optical regime and subsequently in electron microscopy, we present a methodology that is applicable at different spectral regions and robust to non-orthogonality. The distributed illumination pattern sets also result in a reduced peak intensity, thereby potentially reducing damage of samples during imaging. This imaging approach is potentially translatable beyond both regimes explored here, as a single-element detector system.

Deep-3D microscope: 3D volumetric microscopy of thick scattering samples using a wide-field microscope and machine learning.

Confocal microscopy is a standard approach for obtaining volumetric images of a sample with high axial and lateral resolution, especially when dealing with scattering samples. Unfortunately, a confocal microscope is quite expensive compared to traditional microscopes. In addition, the point scanning in confocal microscopy leads to slow imaging speed and photobleaching due to the high dose of laser energy. In this paper, we demonstrate how the advances in machine learning can be exploited to "teach" a traditional wide-field microscope, one that's available in every lab, into producing 3D volumetric images like a confocal microscope. The key idea is to obtain multiple images with different focus settings using a wide-field microscope and use a 3D generative adversarial network (GAN) based neural network to learn the mapping between the blurry low-contrast image stacks obtained using a wide-field microscope and the sharp, high-contrast image stacks obtained using a confocal microscope. After training the network with widefield-confocal stack pairs, the network can reliably and accurately reconstruct 3D volumetric images that rival confocal images in terms of its lateral resolution, z-sectioning and image contrast. Our experimental results demonstrate generalization ability to handle unseen data, stability in the reconstruction results, high spatial resolution even when imaging thick (∼40 microns) highly-scattering samples. We believe that such learning-based microscopes have the potential to bring confocal imaging quality to every lab that has a wide-field microscope.

Two-photon scanned light sheet fluorescence microscopy with axicon imaging for fast volumetric imaging.

.Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition.Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM.Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM.Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz.Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.

Metrological performance analysis of optical coherent tomography

A spectral-domain optical coherence tomography (SD-OCT) system with 1555 nm center wavelength is tested for stability and accuracy from the micron level to the sub-micron level. The energy centrobaric correction method is used to extract the sub-axial-resolution information as the optical path difference, i.e., the topographic height. We compared the used algorithm with the widely applied phase slope method. The stability of a point scanning OCT system in Michelson style is experimentally compared with that of a system in Fizeau style. Several simple and complicated samples are measured and compared. Finally, we compare the topographic performance of the OCT system with a commercial confocal microscope and white-light interferometer (WLI).

Evaluation of the Large-Length Indoor Standard Installation Based on Single Point Scanning

The large length indoor standard installation (LLISI) serves as a standard traceability system for large-scale measuring devices, and hence evaluating the accuracy of its measurements during motion is of great importance. A laser tracker, as a single-point scanning method with strong anti-interference ability and high accuracy for 3D measurement, can meet the measurement requirements of LLISI. In this study, a laser tracker was used to evaluate the accuracy of LLISI during motion based on the single-point scanning method. Fifteen measurement combinations including five measurement intervals and three measurement speeds were selected in the experiment and the results were compared with the measurement data of a laser interferometer. By analyzing the local movement speed of the ISS platform, the uncertainty in local measurement speed of the platform was evaluated. The results showed that the laser tracker has high measurement accuracy and good repeatability for the measurement of LLISI, which can provide strong support for data resource protection of LLISI.

Applying TADF Emitters in Bioimaging and Sensing-A Novel Approach Using Liposomes for Encapsulation and Cellular Uptake.

A new method for facilitating the delivery, uptake and intracellular localisation of thermally activated delayed fluorescence (TADF) complexes was developed. First, confinement of TADF complexes in liposomes was demonstrated, which were subsequently used as the delivery vehicle for cellular uptake. Confocal fluorescence microscopy showed TADF complexes subsequently localise in the cytoplasm of HepG2 cells. The procedures developed in this work included the removal of molecular oxygen in the liposome preparation without disrupting the liposome structures. Time-resolved fluorescence microscopy (point scanning) showed initial prompt fluorescence followed by a weak, but detectable, delayed fluorescence component for liposomal TADF internalised in HepG2 cells. By demonstrating that it is possible to deliver un-functionalised and/or unshielded TADF complexes, a sensing function for TADFs, such as molecular oxygen, can be envisaged.

Characterization of the spectral memory effect of scattering media.

The optical memory effect is an interesting phenomenon exploited for deep-tissue optical imaging. Besides the widely studied memory effects in the spatial domain to accelerate point scanning speed, the spectral memory effect is also important in multispectral wavefront shaping. Although being theoretically analyzed for decades, quantitative studies of spectral memory effect on a variety of scattering media including biological tissue were rarely reported. In practice, quantifying the range of the spectral memory effect is essential in efficiently shaping broadband light, as it determines the optimum spectral resolution in realizing spatiotemporal focus through scattering media. In this work, we analyze the spectral memory effect based on a diffusion model. An explicit analytical expression involves the illumination wavelength, the diffusion constant, and the sample thickness is derived, which is consistent with the one in the literature. We experimentally quantified the range of spectral correlation for two types of biological tissue, tissue-mimicking phantoms with different concentrations, and diffusers. Specifically, for tissue-mimicking phantoms with calibrated scattering parameters, we show that a correction factor of more than 20 should be inserted, indicating that the range of spectral correlation is much larger than one would expect. This finding is particularly beneficial to multispectral wavefront shaping, as stringent requirements on the spectral resolution could be alleviated by at least one order of magnitude.

Low-Cost Multispectral System Design for Pigment Analysis in Works of Art.

To better understand and preserve works of art, knowledge is needed about the pigments used to create the artwork. Various noninvasive techniques have been used previously to create pigment maps, such as combining X-ray fluorescence and hyperspectral imaging data. Unfortunately, most museums have limited funding for the expense of specialized research equipment, such as hyperspectral reflectance imaging systems. However, many museums have hand-held point X-ray fluorescence systems attached to motorized easels for scanning artwork. To assist museums in acquiring data that can produce similar results to that of HSI systems, while minimizing equipment costs, this study designed and modeled a prototype system to demonstrate the expected performance of a low-cost multispectral system that can be attached to existing motorized easels. We show that multispectral systems with a well-chosen set of spectral bands can often produce classification maps with value on par with hyperspectral systems. This study analyzed the potential for capturing data with a point scanning system through predefined filters. By applying the system and noise modeling parameters to HSI data captured from a 14th-Century illumination, the study reveals that the proposed multispectral imaging system is a viable option for this need.

Deep learning-based protocols to enhance infrared imaging systems

Infrared (IR) spectroscopic imaging provides both morphologic and chemical detail; however, obtaining this extensive spectral-spatial information requires the ability to rapidly record high-quality data. Discrete frequency infrared (DFIR) imaging using a point scanning microscope strikes a balance between data quality and acquisition speed that, in principle, can further be aided by computational methods. Here, we report a deep learning-based framework to complement the process of data acquisition and information extraction. First, we introduce a convolutional neural network (CNN) to leverage both spatial and spectral information for segmenting data into informative sub-classes, which we call the IR-SEG network. We show that this framework increases accuracy by using approximately half of the features used in the typical pixel-wise classification of IR data. Second, we present a generative adversarial network (GAN)-based approach to reconstruct full data sets with low loss in the information from incomplete spatial and spectral data recording. Termed IR-REC, this approach is shown to speed up data acquisition by up to 20-fold for typical biomedical samples. In addition to enhancing the speed and quality of data, we also propose a method to utilize complementary morphologic detail to estimate the spatial details of a single band IR image beyond the diffraction limit. Finally, we discuss potential pitfalls and new opportunities that can be addressed by developing these methods further. Together, these deep learning techniques provide new capabilities for IR imaging to extract better quality information faster.

Pupil function design for multifocal confocal, STED, and isoSTED microscopy.

Point scanning super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy are powerful tools to observe biological samples at sub-diffraction limited resolution in three dimensions. However, scanning the sample with only a single beam limits the imaging speed in these microscopes. Here, we propose a concept to increase this speed by introducing highly flexible multifocal illumination and detection. We introduce phase patterns in the objectives’ pupil planes to create arrays of foci in the sample plane with negligible loss of laser power. High uniformity of these foci’s intensities is achieved by iteratively applying a weighted Gerchberg–Saxton phase retrieval algorithm. We characterize the performance of this iterative approach numerically and present simulation results that demonstrate the high quality of the focus arrays for future implementations in laser-scanning STED and isoSTED microscopes. The same approach can also be applied in diffraction-limited confocal laser scanning microscopy.

SPADs and SiPMs Arrays for Long-Range High-Speed Light Detection and Ranging (LiDAR).

Light Detection and Ranging (LiDAR) is a 3D imaging technique, widely used in many applications such as augmented reality, automotive, machine vision, spacecraft navigation and landing. Achieving long-ranges and high-speed, most of all in outdoor applications with strong solar background illumination, are challenging requirements. In the introduction we review different 3D-ranging techniques (stereo-vision, projection with structured light, pulsed-LiDAR, amplitude-modulated continuous-wave LiDAR, frequency-modulated continuous-wave interferometry), illumination schemes (single point and blade scanning, flash-LiDAR) and time-resolved detectors for LiDAR (EM-CCD, I-CCD, APD, SPAD, SiPM). Then, we provide an extensive review of silicon- single photon avalanche diode (SPAD)-based LiDAR detectors (both commercial products and research prototypes) analyzing how each architecture faces the main challenges of LiDAR (i.e., long ranges, centimeter resolution, large field-of-view and high angular resolution, high operation speed, background immunity, eye-safety and multi-camera operation). Recent progresses in 3D stacking technologies provided an important step forward in SPAD array development, allowing to reach smaller pitch, higher pixel count and more complex processing electronics. In the conclusions, we provide some guidelines for the design of next generation SPAD-LiDAR detectors.

Surface Exposure Technology of Metal Powder Based on an Optically-Addressed Liquid Crystal Spatial Light Modulator

Compared with the current mature point scanning metal additive manufacturing technology, the surface exposure technology by laser additive manufacturing can accelerate the construction speed, and the short pulse laser is expected to improve the resolution. Here, a surface exposure technology processing system was constructed based on a nanosecond pulsed laser source and an optically-addressed liquid crystal spatial light modulator with a damage threshold higher than 500 mJ/cm2. The resolution of the surface exposure processing system is proved to be about 100 μm.

Single-scan HiLo with line-illumination strategy for optical section imaging of thick tissues.

Optical sectioning has been widely employed for inhibiting out-of-focus backgrounds in three-dimensional (3D) imaging of biological samples. However, point scanning imaging or multiple acquisitions for wide-field optical sectioning in epi-illumination microscopy remains time-consuming for large-scale imaging. In this paper, we propose a single-scan optical sectioning method based on the hybrid illumination (HiLo) algorithm with a line-illumination strategy. Our method combines HiLo background inhibition with confocal slit detection. It thereby offers a higher optical sectioning capability than wide-field HiLo and line-confocal imaging without extra modulation and multiple data acquisition. To demonstrate the optical-sectioning capability of our system, we imaged a thin fluorescent plane and different fluorescence-labeled mouse tissue. Our method shows an excellent background inhibition in thick tissue and thus potentially provides an alternative tool for 3D imaging of large-scale biological tissue.

Ultrahigh-speed point scanning two-photon microscopy using high dynamic range silicon photomultipliers

Conventional two-photon microscopes use photomultiplier tubes, which enable high sensitivity but can detect relatively few photons per second, forcing longer pixel integration times and limiting maximum imaging rates. We introduce novel detection electronics using silicon photomultipliers that greatly extend dynamic range, enabling more than an order of magnitude increased photon detection rate as compared to state-of-the-art photomultiplier tubes. We demonstrate that this capability can dramatically improve both imaging rates and signal-to-noise ratio (SNR) in two-photon microscopy using human surgical specimens. Finally, to enable wider use of more advanced detection technology, we have formed the OpenSiPM project, which aims to provide open source detector designs for high-speed two-photon and confocal microscopy.

Digital adaptive optics based on digital lateral shearing of the computed pupil field for point scanning retinal swept source OCT

A novel non-iterative digital adaptive optics technique is presented in which the wavefront error is calculated using the phase difference between the pupil field and its digital copies translated by a pixel along the horizontal and vertical direction in the pupil plane. This method provides slope data per pixel, thus can generate > 50k local slope data samples for a circular pupil of diameter 256 pixels with high accuracy and dynamic range. It offers more than 12x faster computational speed in comparison to the sub-aperture based digital adaptive optics method. Furthermore, it is independent of any system parameters, the light distribution in the pupil plane, or the intensity of the image. The technique is useful in applications such as interferometric or digital holography based microscopy, metrology, and as digital wavefront sensor in adaptive optics, where focusing of light in the sample is involved that creates a guide star or where the sample itself exhibits guide star-like structures. This technique is implemented in a point scanning swept-source OCT at 1060 nm, and a large wavefront error with a peak to valley of 20 radians and root mean square error of 0.71 waves is detected and corrected in case of a micro-beads phantom sample. Also, human photoreceptor images are recovered from aberrated retinal OCT volumes acquired at eccentricities of 2 and 2.5 degrees from the fovea in vivo.

The influence of the high potassium biomass on the ash fusion characteristics of coal

The influence of high potassium biomass (corn straw (CS)) on ash fusion temperature (AFTs) of Jincheng coal (JC) was explored by X-ray fluorescence (XRF), intelligent ash fusion point tester, X-ray diffraction (XRD), and scanning electron microscopy-energy dispersive spectrometer (SEM-EDS). The results show that the method of adding CS can effectively reduce the AFTs of JC. The main mineral composition of JC ash at high temperatures is refractory Al6Si2O13, which is why JC has a higher AFTs; the main mineral composition of CS ash at high temperatures is its ash with a large amount of Ca/Mg-containing aluminosilicates and glassy substance containing metal potassium (K), which is why CS has a lower AFTs. With the increasing of the proportion of CS added, Al6Si2O13 as the main mineral component in JC ash gradually converts into a series of new Ca/K-containing aluminosilicates (such as, diopside (CaMgSi2O6), leucite (KAlSi2O6), etc.), which results in the reduction of the AFTs of JC. Meanwhile, the Ca/K-containing aluminosilicates has a low-temperature co-fusion effect. In addition, SiO2 and Al2O3 together in coal ash have excellent K-fixing efficiency confirmed by the formation of stable K-containing aluminosilicates.