Conventional Illumination Research Articles

Spatial frequency modulated imaging in coherent anti-Stokes Raman microscopy.

For sparse samples or in the presence of ambient light, the signal-to-noise ratio (SNR) performance of single-point-scanning coherent anti-Stokes Raman scattering (CARS) images is not optimized. As an improvement, we propose replacing the conventional CARS focus-point illumination with a periodically structured focus line while continuing to collect the transmitted CARS intensity on a single detector. The object information along the illuminated line is obtained by numerically processing the CARS signal recorded for various periods of the structured focus line. We demonstrate experimentally the feasibility of this spatial frequency modulated imaging (SPIFI) in CARS (SPIFI-CARS) and SHG (SPIFI-SHG) and identify situations where its SNR is better than that of the single-point-scanning approach.

Configuration design for the variable pupil shaping unit of a photolithography machine.

This work provides an integrated design and simulation method for a pupil shaping unit, which combines optical design and simulation software using the Python programming language. The novel approach presented here allows for systematic optimization and solves the inverse problem to obtain the configuration of a pupil shaping unit with given partial coherence factors (sigmas). The relationships among sigma, focal length, zoom lens separation, axicon separation, and ring width are investigated for conventional and annular illumination modes. Results of the pupil shaping unit for a NA 0.75 193 nm photolithography machine are presented, and a new interpolation table design method that simplifies the original method is proposed. The maximum residual experimental error for sigma is 0.01106.

Principles of co‐axial illumination for photochemical reactions: Part 1. Model development

AbstractPhotochemical reactors with conventional homogeneous illumination suffer from a light efficiency problem, which is inherent to their design: Dark zones arise near the reagent‐rich inlet whereas the reagent depleted outlet is over‐illuminated. Any attempt to mitigate dark zones at the inlet will only increase photon losses further downstream. This study reports the principles and model equations for co‐ and counter‐current illumination in photochemical reactors, along with an optimization study to determine the most efficient and productive operating point. This work proves that the use of co‐ and counter‐current illuminated reactors increases the energy efficiency while easing scalability by implementing larger path lengths, without altering the reactor's geometry. We report a simple model to determine the conversion obtained by such novel illumination techniques and compare it to the current state‐of‐the‐art. Two nondimensional groups where derived that describe all possible reactor configurations, these are the initial absorbance (A) and the quantum photon balance (ρϕ). Variation of both parameters leads for noncompetitive photochemical reactions to an optimal point for the current state‐of‐the‐art as well as the novel co‐axial illumination. Ultimately, we recommend the use of an initial absorbance value (A) of at least 1, and a quantum photon balance (ρϕ) equal to 1 to introduce sufficient light and enable near complete absorption of light.

Rapid and Extreme Low-light Superresolution Imaging via Artificial Intelligence

Supreresolution (SR) fluorescence microscopy breaks the optical diffraction limit and achieves unprecedented nanoscale resolution, thus producing a huge impact on biological research. Structured illumination microscopy (SIM) is one of the most broadly used SR techniques. SIM applies varying, nonuniform illumination to samples. A dedicated computational algorithm is then used to retrieve SR information from nine or fifteen sequentially acquired images, for 2D or 2D/3D reconstruction respectively. Super-resolution radial fluctuation (SRRF) is a purely computational SR approach, that can retrieve high frequency information by spatio-temporally analyzing the stochastically fluctuating signals from image time series (typically >200 frames). We utilized deep neural networks to directly reconstruct super-resolved images out of a series of images taken with either structured or conventional wide-field illumination microscopy. This substantially reduced the number of images compared to state-of-the-art methods (as few as 3 images for SIM and 5 images for SRRF). Further, leveraging skip-layer connected U-Nets, we could restore high quality, high-resolution images from raw data with over 100 times less photons. We validated our results for different cellular structures, including microtubules, mitochondria, adhesions and actin filaments. We demonstrated its applications in ultra-fast, multi-color superresolution imaging of living cells. A ready-to-use open source package is available.

Energy optimization of a light projection system for buildings that virtually restores artworks

The need to achieve energy efficiency standards in the lighting systems of buildings makes it necessary to optimize all aspects of them. Here, the development of a light projection system that achieves this goal by studying and modifying the spectral output, compared to conventional illumination, is described. A lighting system that estimates the reflectance characteristics of artwork and emits optimized lighting can reduce light absorption. A damage-minimizing point-by-point light projection system is developed using an optimization algorithm, to improve the appearance of the surfaces of artworks whose color has faded. In this case, a simulation of an aged oil painting was made by manipulating the original photograph, which was printed and to which the proposed system was applied. The results show that, when the aged printed image is illuminated with the optimized light source, it appears indistinguishable from the non-aged oil painting.

Multi-lobe superoscillation and its application to structured illumination microscopy.

Superoscillating function is a band-limited function that is locally oscillating faster than its highest Fourier component. In this work, we study and implement methods to generate multi-lobe optical superoscillating beams, with nearly constant intensity and constant local frequency. We generated superoscillating patterns having up to 12 sub-wavelength oscillations, with local frequency of 20% to 40% above the band-limit. We then test the potential application of these beams to super-resolution structured illumination microscopy. By utilizing the Moiré effect on a fluorescent grating, we have demonstrated experimentally resolution improvement over the conventional sinusoidal illumination. Our simulations show that structured illumination microscopy with super oscillating multi-lobe beams can provide more than twofold improvement in resolution, with respect to the classical diffraction limit and for coherent or incoherent modalities.

A regimen to minimize pain during blue light photodynamic therapy of actinic keratoses: Bilaterally controlled, randomized trial of simultaneous versus conventional illumination

Bluelight photodynamic therapy (PDT) is effective for actinic keratosis, but many patients experience stinging pain during illumination. To compare a conventional regimen (1hour of 5-aminolevulinic acid [ALA] preincubation, followed by blue light) versus a new modified regimen in which blue light is started immediately after ALA application. A clinical trial with a bilaterally controlled, intrapatient study design was conducted with 23 patients. Topical 20% ALA was applied to the entire face and/or scalp. On 1 side of the body, blue light was started immediately and continued for either 30, 45, or 60minutes (simultaneous PDT). On the contralateral side, the blue light began 1hour after ALA application and lasted 1000seconds (conventional PDT). Pain was evaluated on a scale from 0 to 10. Actinic keratosis lesion counts were determined by clinical examination and photography. All patients experienced significantly less pain during simultaneous illumination than during the conventional regimen. At 3months after treatment, lesion clearance was nearly identical on the 2 sides, as determined by statistical testing of noninferiority ± 15% margin. Although bilaterally controlled, the study was relatively small. Additional studies are recommended. The modified PDT regimen is essentially painless, yet it provides treatment efficacy similar to a conventional regimen.

Optimal illumination scheme for isotropic quantitative differential phase contrast microscopy

Differential phase contrast microscopy (DPC) provides high-resolution quantitative phase distribution of thin transparent samples under multi-axis asymmetric illuminations. Typically, illumination in DPC microscopic systems is designed with two-axis half-circle amplitude patterns, which, however, result in a non-isotropic phase contrast transfer function (PTF). Efforts have been made to achieve isotropic DPC by replacing the conventional half-circle illumination aperture with radially asymmetric patterns with three-axis illumination or gradient amplitude patterns with two-axis illumination. Nevertheless, the underlying theoretical mechanism of isotropic PTF has not been explored, and thus, the optimal illumination scheme cannot be determined. Furthermore, the frequency responses of the PTFs under these engineered illuminations have not been fully optimized, leading to suboptimal phase contrast and signal-to-noise ratio for phase reconstruction. In this paper, we provide a rigorous theoretical analysis about the necessary and sufficient conditions for DPC to achieve isotropic PTF. In addition, we derive the optimal illumination scheme to maximize the frequency response for both low and high frequencies (from 0 to 2NAobj) and meanwhile achieve perfectly isotropic PTF with only two-axis intensity measurements. We present the derivation, implementation, simulation, and experimental results demonstrating the superiority of our method over existing illumination schemes in both the phase reconstruction accuracy and noise-robustness.

Single-pixel imaging of the retina through scattering media.

Imaging the retina of cataractous patients is useful to detect pathologies before the cataract surgery is performed. However, for conventional ophthalmoscopes, opacifications convert the lens into a scattering medium that may greatly deteriorate the retinal image. In this paper we show, as a proof of concept, that it is possible to surpass the limitations imposed by scattering applying to both, a model and a healthy eye, a newly developed ophthalmoscope based on single-pixel imaging. To this end, an instrument was built that incorporates two imaging modalities: conventional flood illumination and single-pixel based. Images of the retina were acquired firstly in an artificial eye and later in healthy living eyes with different elements which replicate the scattering produced by cataractous lenses. Comparison between both types of imaging modalities shows that, under high levels of scattering, the single-pixel ophthalmoscope outperforms standard imaging methods.

Maximum advantage of quantum illumination

Discriminating between quantum states is a fundamental problem in quantum information protocols. The optimum approach saturates the Helstrom bound, which quantifies the unavoidable error probability of mistaking one state for another. Computing the error probability directly requires complete knowledge and diagonalization of the density matrices describing these states. Both of these fundamental requirements become impractically difficult to obtain as the dimension of the states grow large. In this article, we analyze quantum illumination as a quantum channel discrimination protocol and circumvent these issues by using the normalized Hilbert-Schmidt inner product as a measure of distinguishability. Using this measure, we show that the greatest advantage gained by quantum illumination over conventional illumination occurs when one uses a Bell state.

Fast structured illumination microscopy with reflectance disturbance resistibility and improved accuracy.

The conventional fast structured illumination microscopy (FSIM) without vertical scanning has been proposed by Lee for fast surface profiling. Nevertheless, this method is vulnerable to nonuniform surface reflectivity which is generally caused by the different reflection characteristic of hybrid materials and the environmental fluctuations. Consequently, traditional IFSIM cannot be used in the profile measurement for the samples with surface heterogeneity. In this paper, an improved FSIM configuration (IFSIM) is explored for improving the axial accuracy and suppressing the disturbance of the reflectivity. With this technique, a sinusoidal pattern produced by the digital micro-mirror device (DMD) is illuminated onto the sample. The reflected patterns are captured by two charge-coupled devices (CCDs) that are separately placed in and behind or before the focal plane. The subtraction and sum values of the two axial modulation response curves (AMRs) are divided as the new response function, which effectively suppresses the influence of the inhomogeneous reflectivity. Both wide dynamic range and enhanced axial accuracy can be selectively obtained by controlling the defocusing amount of the two differential detectors. The theoretical analysis and experiments are conducted to verify the feasibility of this method.

Profilometry With Enhanced Accuracy Using Differential Structured Illumination Microscopy

The conventional structured illumination micro-scopy (SIM) reconstructs the surface through determining the focal position from the modulation depth response (MDR) using the Gaussian fitting. In this letter, an innovative differential structured illumination microscopy (DSIM) is developed. This technique exploits the position at the linear portion of the MDR to achieve the measurements. To extract this position, two detectors are used to generate the differential modulation depth response (DMDR). After that, the zero point of the DMDR for each pixel is extracted by the linear fitting technique. The experiments and theoretical analysis are elaborated to verify that the DSIM can greatly enhance the axial accuracy, providing that the potential to be applied in high-precision measurement.

Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is ≥80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

Structured illumination in compact and field-portable 3D-printed shearing digital holographic microscopy for resolution enhancement.

A compact and field-portable three-dimensional (3D)-printed structured illumination (SI) digital holographic microscope based on shearing geometry is presented. By illuminating the sample using a SI pattern, the lateral resolution in both reconstructed phase and amplitude images can be improved up to twice the resolution provided by conventional illumination. The use of a 3D-printed system and shearing geometry reduces the complexity of the system, while providing high temporal stability. The experimental results for the USAF resolution target show a resolution improvement of a factor of two which corroborates the theoretical prediction. Resolution enhancement in phase imaging is also demonstrated by imaging a biological sample. To the best of our knowledge, this is the first report of a compact and field-portable SI digital holographic system based on shearing geometry.

The impact of medium composition and photosynthetically active radiation level on the initial in vitro growth and production of flavonoids of Vernonia condensata Baker

Abstract The use of techniques such as micropropagation can enable studying the chemical components of species with medicinal potential. However, the photosynthetically active radiation (PAR) supplied by the conventional illumination systems used for in vitro cultures is typically very low. The objective of this work was to evaluate the effect of variation of the sucrose content of the culture medium and the PAR level on the in vitro growth of Vernonia condensata, as well as to obtain the flavonoid profile of this species. Micropropagated shoots were inoculated in MS medium with half the normal saline concentration, supplemented with 15 g L−1 or 30 g L−1 of sucrose and 7 g L−1 of agar, and were maintained under three PAR levels: 40, 80 or 120 μmol m−2 s−1. Samples of the aerial part and root system of the plants subjected to the respective treatments were dried for quantitative analysis of the flavonoids by applying high-performance liquid chromatography (HPLC) to the hydromethanolic extract, with programming of the elution gradient. The HPLC method was validated according to the parameters specificity, linearity, precision, accuracy, limit of detection and limit of quantification. Rutin and quercetin 3-β-D-glucoside were the main flavonoids produced by this species when grown in vitro, with the aerial part producing more than the roots according to the quantification method used. The results demonstrate the influence of sucrose concentration and PAR intensity on the in vitro development and flavonoid profile of V. condensata.

Corrigendum: Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy.

[This corrects the article DOI: 10.3389/fncel.2018.00315.].

Structured-illumination reflectance imaging for the detection of defects in fruit: Analysis of resolution, contrast and depth-resolving features

Structured-illumination reflectance imaging (SIRI), compared to conventional uniform illumination based modalities, provides a new means for defect detection of fruit. The capability of SIRI for defect detection, however, mainly depends on its image resolution and contrast and depth-resolving features. This study was therefore aimed at providing theoretical and experimental analyses of these features for SIRI, so as to evaluate its potential for detecting surface and subsurface defects of fruit and other food products. The image formation in SIRI was first modelled in terms of the optical transfer functions, which led to a general image demodulation methodology and also provided insights into the features of direct component (DC) and amplitude component (AC) images that were demodulated from the original SIRI pattern images. A set of experiments were performed on standard optical targets and apple samples by using illumination patterns over a wide range of spatial frequencies of 0.01–1.0 cycles mm−1 to examine the features of SIRI. The imaging process acted as a low-pass filter, which, coupled with the sample absorption and scattering, accounted for the resolution and contrast loss of resulting images. As the hallmark of SIRI, AC images possessed enhanced resolution and contrast, which could be explained by the synergistic effect of frequency shifting in the Fourier domain and suppressed light scattering in the sample, and also a depth-resolved ability by varying the spatial frequency of illumination patterns. However, the depth of tissue interrogation by the AC images decreased as the spatial frequency of illumination patterns increased. The effectiveness of AC in defect detection of apples depended on such factors as defect type, fruit surface morphology (thus variety) and spatial frequency of illumination. Without resorting to an inverse computational approach, SIRI should be positioned as a subsurface-surface modality for detecting defects that are on or close to the surface of fruits, especially thin-skinned fruits like apple. Further research is therefore needed to explore the full potential of SIRI in defect detection of fruits and other food products.

A multi-mode digital holographic microscope.

We present a transmission-mode digital holographic microscope that can switch easily between three different imaging modes: inline, dark field off-axis, and bright field off-axis. Our instrument can be used: to track through time in three dimensions microscopic dielectric objects, such as motile micro-organisms; localize brightly scattering nanoparticles, which cannot be seen under conventional bright field illumination; and recover topographic information and measure the refractive index and dry mass of samples via quantitative phase recovery. Holograms are captured on a digital camera capable of high-speed video recording of up to 2000 frames per second. The inline mode of operation can be easily configurable to a large range of magnifications. We demonstrate the efficacy of the inline mode in tracking motile bacteria in three dimensions in a 160 μm × 160 μm × 100 μm volume at 45× magnification. Through the use of a novel physical mask in a conjugate Fourier plane in the imaging path, we use our microscope for high magnification, dark field off-axis holography, demonstrated by localizing 100 nm gold nanoparticles at 225× magnification up to at least 16 μm from the imaging plane. Finally, the bright field off-axis mode facilitates quantitative phase microscopy, which we employ to measure the refractive index of a standard resolution test target and to measure the dry mass of human erythrocytes.

A Versatile and Open-Source Rapid LED Switching System for One-Photon Imaging and Photo-Activation.

Combining fluorescence and transmitted light sources for microscopy is an invaluable method in cellular neuroscience to probe the molecular and cellular mechanisms of cells. This approach enables the targeted recording from fluorescent reporter protein expressing neurons or glial cells in brain slices and fluorescence-assisted electrophysiological recordings from subcellular structures. However, the existing tools to mix multiple light sources in one-photon microscopy are limited. Here, we present the development of several microcontroller devices that provide temporal and intensity control of light emitting diodes (LEDs) for computer controlled microscopy illumination. We interfaced one microcontroller with μManager for rapid and dynamic overlay of transmitted and fluorescent images. Moreover, on the basis of this illumination system we implemented an electronic circuit to combine two pulsed LED light sources for fast (up to 1 kHz) ratiometric calcium (Ca2+) imaging. This microcontroller enabled the calibration of intracellular Ca2+ concentration and furthermore the combination of Ca2+ imaging with optogenetic activation. The devices are based on affordable components and open-source hardware and software. Integration into existing bright-field microscope systems will take ∼1 day. The microcontroller based LED imaging substantially advances conventional illumination methods by limiting light exposure and adding versatility and speed.

Experimental Demonstration of Hyperbolic Metamaterial Assisted Illumination Nanoscopy

An optical metamaterial is capable of manipulating light in nanometer scale that goes beyond what is possible with conventional materials. Taking advantage of this special property, metamaterial-assisted illumination nanoscopy (MAIN) possesses tremendous potential to extend the resolution far beyond conventional structured illumination microscopy. Among the available MAIN designs, hyperstructured illumination that utilizes strong dispersion of a hyperbolic metamaterial (HMM) is one of the most promising and practical approaches, but it is only theoretically studied. In this paper, we experimentally demonstrate the concept of hyperstructured illumination. A ∼80 nm resolution has been achieved in a well-known Ag/SiO2 multilayer HMM system by using a low numerical aperture objective (NA = 0.5), representing a 6-fold resolution enhancement of the diffraction limit. The resolution can be significantly improved by further material optimization.