Construction of an instant structured illumination microscope

Pixel super-resolution (PSR) techniques have been developed to overcome the sampling limit in lensless digital holographic imaging. However, the inherent non-convexity of the PSR phase retrieval problem can potentially degrade reconstruction quality by causing the iterations to tend toward a false local minimum. Furthermore, the ill posedness of the up-sampling procedure renders PSR algorithms highly susceptible to noise. In this Letter, we propose a heuristic PSR algorithm with adaptive smoothing (AS-PSR) to achieve high-fidelity reconstruction. By automatically adjusting the intensity constraints on the estimated field, the algorithm can effectively locate the optimal solution and converge with high reconstruction quality, pushing the resolution toward the diffraction limit. The proposed method is verified experimentally within a coherent modulation phase retrieval framework, achieving a twofold improvement in resolution. The AS-PSR algorithm can be further applied to other phase retrieval methods based on alternating projection.

Design and calibration of spreading equipment for agricultural materials are critically important but is facing some limitations in time and costs. Lack of personnel and environmental constraints, as well as equipment features present challenges in the calibration process and results. The major objective of this project was to introduce and investigate the feasibility of integration of automated electrical equipment into this calibration process in order to optimize the overall procedure in terms of cost, time and precision as an ultimate goal. Specific objectives focused on optical techniques as well as novel accelerometer technology. A photo based method in detection of collected grains based on counting of particles was investigated. Results from tests with three different grain particles showed that the sensor could be used in applications up to 20gr of collected materials in one pass. The sensor could detect multiple particles’ passage at the same time, but was unable to go further than a certain sample weight of 20, 30 and 60 grams for ammonium sulfate, rice and urea respectively while maintaining acceptable precision. In this regard, physical characteristics of the mentioned granular particles were also investigated to better address the importance of physical uniformity in this sensing system. A novel set of sensing plates based on accelerometer technology were introduced and the practicality of the sensors was tested using the standard setup for measurement of spreading patterns. A comparison between the outputs of a set of identical accelerometer plates showed a close estimate of the trend which could be achieved in the same deposition using the traditional dry calibration process. Moreover, the introduced sensor was highly flexible and could be adjusted in various ways in order to better match the application demands. In this regard, the proposed sensor was also tested at 10 and 20 degree angles with respect to the horizon and the results indicated almost twofold improvement in resolution from 595mV/g to 955mV/g at 10 and 20 degrees respectively. This novel accelerometer based technique may be practical for improved automation of calibration techniques.

Astrophysical studies require accurate, sensitive and fast detectors to detect faint sources with high variability. Recently an array of Single Photon Avalanche Diodes (SPAD), SPADA, has been developed. This array is suitable for competitive adaptive optics operations and fast transient image acquisition at a fraction of the current cost of imaging arrays. The fabricated solid-state photon counters are rugged, easily integrated with the optics, free from readout noise, and have very fast frame rates (> 10 kHz, for visible corrections) with nanosecond electronic gating. In this paper, the following are described: the development of silicon monolithic arrays of 60 photon-counters, the detection electronics (based on integrated active quenching circuits for each pixel of the array), the real-time data-processing board implemented into FPGA and some aspects of the mechanical housing.

We propose a novel, to the best of our knowledge, super-resolution technique, namely saturable absorption assisted nonlinear structured illumination microscopy (SAN-SIM), by exploring the saturable absorption property of a material. In the proposed technique, the incident sinusoidal excitation is converted into a nonlinear illumination by propagating through a saturable absorbing material. The effective nonlinear illumination possesses higher harmonics which multiply fold high frequency components within the passband and hence offers more than two-fold resolution improvement over the diffraction limit. The theoretical background of the technique is presented, supported by the numerical results. The simulation is performed for both symmetric as well as random samples where the raw moiré frames are processed through a blind reconstruction approach developed for the nonlinear SIM. The results demonstrate the super-resolution capability of the proposed technique.

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 μm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

Structured illumination microscopy (SIM) is an essential super-resolution microscopy technique that enhances resolution. Several images are required to reconstruct a super-resolution image. However, linear SIM resolution enhancement can only increase the spatial resolution of microscopy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function. The frequency of the pattern generated by the nonlinear response in samples is not limited; therefore, nonlinear SIM (NL-SIM), in theory, has no inherent limit to the resolution. In the present study, we describe a two-photon nonlinear SIM (2P-SIM) technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern, which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process. The theoretical models of super-resolution imaging were established through our simulation, which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the resolution. The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.

We present a low-cost 3D-printing method of fabricating optical quality lenslet arrays for integration in a multifocal structured illumination microscope (mSIM), achieving super-resolution fluorescence imaging using 3D-printed optics for the first time. We detail the design and manufacturing processes to produce high-quality 3D-printed optics, showing their comparable surface roughness of 30 ± 2.5 nm for the 3D-printed elements compared to 37 ± 1.4 nm for commercial glass optics. A 3D-printed lenslet array with a ‘honeycomb’ geometry and 1.2 mm lenslet diameter was compared to a high-end glass commercial lenslet array with 250 µm lenslet diameter and a lower cost commercial lenslet array with a 1 mm by 1.4 mm lenslet footprint. The imaging performance of the different optics was benchmarked using a custom mSIM setup by quantifying the beam profile homogeneity and the experimental lateral resolution. The mSIM setup incorporating the different microlens arrays was tested using a commercial bovine pulmonary artery endothelial cell specimen, highlighting an achievable resolution enhancement from 237 nm ± 12 nm with laser-scanning illumination to 151 ± 12 nm using the high-end commercial microlens array and from 232 ± 18 nm with laser-scanning illumination to 151 nm ± 12 nm using the 3D-printed honeycomb lenslet array. Advantages of improved background rejection through the custom lenslet geometry are discussed, highlighting the super-resolution microscope performance achievable using custom 3D-printed optics costing as low as £0.50 to produce.

Standardization and calibration of optical microscopy systems have become an important issue owing to the increasing role of biological imaging in high-content screening technology. The proper interpretation of data from high-content screening imaging experiments requires detailed information about the capabilities of the systems, including their available dynamic range, sensitivity and noise. Currently available techniques for calibration and standardization of digital microscopes commonly used in cell biology laboratories provide an estimation of stability and measurement precision (noise) of an imaging system at a single level of signal intensity. In addition, only the total noise level, not its characteristics (spectrum), is measured. We propose a novel technique for estimation of temporal variability of signal and noise in microscopic imaging. The method requires registration of a time series of images of any stationary biological specimen. The subsequent analysis involves a multi-step process, which separates monotonic, periodic and random components of every pixel intensity change in time. The technique allows simultaneous determination of dark, photonic and multiplicative components of noise present in biological measurements. Consequently, a respective confidence interval (noise level) is obtained for each level of signal. The technique is validated using test sets of biological images with known signal and noise characteristics. The method is also applied to assess uncertainty of measurement obtained with two CCD cameras in a wide-field microscope.

I5S: Wide-Field Light Microscopy with 100-nm-Scale Resolution in Three Dimensions

In this chapter we present the main concepts and recent advances in the application of semiconductor quantum dots (QD) as agents in bioimaging. We analyze the biologically relevant properties of QDs focusing on the following topics: QD surface treatment and stability, labeling of cellular structures and receptors with QDs, incorporation of QDs in living cells, cytotoxicity of QDs and the influence of the biological environment on the biological and optical properties of QDs. Initially, we consider utilization of QDs as agents in high-resolution bioimaging techniques that can provide information at the molecular level. The diverse range of modern live-cell QD-based imaging techniques with resolution far beyond the diffraction limit of light has been examined. For each technique, we discuss the pros and cons of QD use and deliberate how QDs can be further engineered to facilitate their applications in the respective imaging techniques and to produce significant improvements in resolution. Finally, this chapter provides an analysis of the future prospects for the application of QDs in bioimaging and related areas of biology.

While widefield microscopy has long been constrained by out-of-focus scattering, advancements have generated a solution in the form of confocal laser scanning microscopy (cLSM) and optical sectioning microscopy using structured illumination (OSM). In this study, we aim to investigate, using microglia branching, if cLSM and OSM can produce images with comparable morphological characteristics. By imaging the somatosensory microglia from a tissue slice of a 3-week-old mouse and establishing morphological parameters that characterizes the microglial branching pattern, we were able to show that there is no difference in total length of the branch tree, number of branches, mean branch length and number of primary to terminal branches. We did find that area-based parameters such as mean occupied area and mean surveillance area were bigger in cLSM isolated microglia compared to OSM ones. Additionally, by investigating the difference in acquisition time between techniques and personal costs we were able to establish that the amortization could be made in 6.11 ± 2.93 years in the case of countries with a Human Development Index (HDI) = 7-9 and 7.06 ± 3.13 years, respectably, for countries with HDI < 7. As such, OSM systems seem a valid option if one just wants basic histological evaluation, and cLSM should be considered for groups that demand higher resolution or volumetric images.

Because of the fingerprint-like specificity of its characteristic spectrogram, Raman spectral imaging has been applied widely in various research areas. Using a combination of structured illumination with the surface-enhanced Raman scattering (SERS) technique, wide-field Raman imaging is developed with a significant improvement in spatial resolution. As a result of the relatively narrow Raman characteristic peaks, optically encoded SERS nanoparticles can be used to perform multiplexed imaging. The results show excellent superresolution wide-field multiplexed imaging performance. The developed technique has extraordinary potential for applications in biological imaging and other related fields.

Because structured illumination microscopy (SIM) has the advantages of wide-field, rapid imaging, and biocompatibility, it is widely used for super-resolution imaging of living cells. Here, we demonstrate a new super-resolution imaging method—bulk plasmon polariton based SIM (BPPSIM), which combines the SIM with the hyperbolic metamaterials (HMMs). By utilizing an HMM composed of a multilayer metal/dielectric film, a deep subwavelength bulk plasmon polariton is obtained. The imaging resolution of BPPSIM can be improved by the uniform and wide-field structured illumination pattern with high spatial frequency, benefiting from the filtering effect of HMM for the spatial frequency spectrum. The resolution of the recovered images by BPPSIM has been increased to 1/8 of the fluorescence wavelength, which is 2.7-fold enhancement in resolution compared with traditional fluorescence microscopy. This proposed approach demonstrates the operability and flexibility of the structured illumination pattern and can be used in a simple, wide-field and super-resolution fluorescence microscope.

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence.

Until very recently, super-resolution localization and tracking of fluorescent particles used camera-based wide-field imaging with uniform illumination. Then it was demonstrated that structured illuminations encode additional localization information in images. The first demonstration of this uses scanning and hence suffers from limited throughput. This limitation was mitigated by fusing camera-based localization with wide-field structured illumination. Current implementations, however, use effectively only half the localization information that they encode in images. Here we demonstrate how all of this information may be exploited by careful calibration of the structured illumination. Our approach achieves maximal resolution for given structured illumination, has a simple data analysis, and applies to any structured illumination in principle. We demonstrate this with an only slightly modified wide-field microscope. Our protocol should boost the emerging field of high-precision localization with structured illumination.