Illumination Scheme Research Articles

Homogeneous large field-of-view and compact iSCAT-TIRF setup for dynamic single molecule measurements

Interferometric scattering microscopy (iSCAT) enables prolonged and high frame rate single particle tracking (SPT) for studying molecular dynamics. Typical iSCAT setups employ conventional widefield or scanning illumination schemes. However, these implementations limit the field-of-view (FoV), the uniformity of the illumination and thus comparable accuracy over the whole FoV, and/or the maximum sampling rate, while in parts increasing hardware requirements and setup size. We demonstrate the realization of a large (60 µm x 60 µm) uniformly illuminated FoV through a passive refractive optical element in the iSCAT illumination path. This scanning-free iSCAT microscope setup is further combined with an objective based total internal reflection fluorescence microscopy (TIRF) channel for a complementary fluorescence readout, a focus-lock system, and a tailored control platform via the open-source ImSwitch software, and it has a compact footprint. As a proof-of-principle, we highlight the performance of the setup through the acquisition of iSCAT images with a uniform contrast and a constant ≤10 nm localization precision throughout the whole FoV. The performance is further demonstrated through dynamic iSCAT SPT and imaging fluorescence correlation spectroscopy (imaging FCS) of lipid diffusion in a model membrane system, highlighting the ability to track a large number of molecules with the same accuracy over a large FoV. Our iSCAT setup thus depicts an accurate and improved way of recording fast molecular dynamics in life sciences.

Optimized galvanometric illumination for terahertz full-field imaging and computed tomography

The pursuit of high-resolution, high-fidelity, real-time imaging is receiving significant attention in terahertz community. In this study, a versatile illumination approach based on a double-mirror galvanometer is proposed and optimized for multiple terahertz imaging approaches. We analyzed the mechanism of galvanometric illumination and elucidated two main factors affecting its homogeneity and parallelism properties. In our module, the terahertz beam is deflected rapidly by the galvanometer which is driven by triangular voltage signals, and then focused by a self-designed aspherical f-θ lens to illuminate the object at an equal lateral scanning velocity. The object beam of different transients is periodically superimposed along the Lissajous trajectory, and is recorded by an array microbolometer in a single integration of 3 s. A homogeneous illumination field is realized with a speckle contrast of 0.11, and the resultant image achieves isotropic resolution. By adopting the proposed galvanometric illumination strategy, the average intensity of the illumination field is increased by 135% compared to expanded coherent illumination, and the speckle contrast is reduced by 83.5% compared to other galvanometric illumination. By virtue of leading low speckle noise, illumination homogeneity and parallelism, a compact imaging system is built for terahertz full-field imaging and computed tomography achieving high imaging speed and fidelity. As a successful attestation of terahertz beam steering, this study is very promising to reduce the total cost, increase the performance and expand the application of terahertz imaging.

Raman spectroscopic quantification of polyethylene particles in water using polydimethylsiloxane-coated nickel foam as a particle-capturing platform

Nickel foam (NF) was evaluated as a medium for the capture of polyethylene (PE) particles in water. NF is a hydrophobic and porous material with a large surface area, making it a promising candidate for attracting PE particles. However, the particle-capturing efficiency using bare NF was only 69.5%. To increase capturing efficiency, a circular polydimethylsiloxane (PDMS)-coated NF (PDMS@NF, diameter: 6 mm) was employed to enhance the hydrophobicity. The capturing efficiency using the PDMS@NF was substantially increased to 97.6 % owing to the increase in hydrophobicity. To quantify the captured PE particles on/in the PDMS@NF using Raman spectroscopy, a wide area illumination (WAI) scheme providing 6 mm-diameter laser illumination was adopted to fully cover the PDMS@NF for representative spectroscopic sampling and accurate quantification. The intensity ratios of PE to PDMS peaks in the collected spectra clearly increased with the quantity of dispersed PE particles (0.1 ∼ 4.0 mg range, R2: 0.992) in the water samples, and the limit of detection was 0.08 mg. Moreover, the capturing efficiencies for polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PET) particles (1 mg of each) using the PDMS@NF were also superior, ranging from 96.4 to 98.2 %. Therefore, the proposed scheme incorporating the PDMS@NF as a particle-capturing and Raman measurement platform has potential as a method for on-line detection of microplastics in water.

Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles.

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity; however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7min over ten 88×88µm2 fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection and EV sizing. We achieve an EV sizing range from 37 to ∼220nm in diameter with a dual 440 and 740nm SP illumination scheme, and suggest that this range can be extended by more advanced image analysis or additional hardware customization. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFI's potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100nm-in-diameter EVs with fluorescently tagged membrane proteins.

Diagonal illumination scheme for Fourier ptychographic microscopy: resolution doubling and aliasing minimization

Diagonal illumination scheme for Fourier ptychographic microscopy: resolution doubling and aliasing minimization

Applications of Lightsheet Fluorescence Microscopy by High Numerical Aperture Detection Lens.

This Review explores the evolution, improvements, and recent applications of Light Sheet Fluorescence Microscopy (LSFM) in biological research using a high numerical aperture detection objective (lens) for imaging subcellular structures. The Review begins with an overview of the development of LSFM, tracing its evolution from its inception to its current state and emphasizing key milestones and technological advancements over the years. Subsequently, we will discuss various improvements of LSFM techniques, covering advancements in hardware such as illumination strategies, optical designs, and sample preparation methods that have enhanced imaging capabilities and resolution. The advancements in data acquisition and processing are also included, which provides a brief overview of the recent development of artificial intelligence. Fluorescence probes that were commonly used in LSFM will be highlighted, together with some insights regarding the selection of potential probe candidates for future LSFM development. Furthermore, we also discuss recent advances in the application of LSFM with a focus on high numerical aperture detection objectives for various biological studies. For sample preparation techniques, there are discussions regarding fluorescence probe selection, tissue clearing protocols, and some insights into expansion microscopy. Integrated setups such as adaptive optics, single objective modification, and microfluidics will also be some of the key discussion points in this Review. We hope that this comprehensive Review will provide a holistic perspective on the historical development, technical enhancements, and cutting-edge applications of LSFM, showcasing its pivotal role and future potential in advancing biological research.

Medium-adaptive compressive diffuse optical tomography.

The low spatial resolution of diffuse optical tomography (DOT) has motivated the development of high-density DOT systems utilizing spatially-encoded illumination and detection strategies. Data compression methods, through the application of Fourier or Hadamard patterns, have been commonly explored for both illumination and detection but were largely limited to pre-determined patterns regardless of imaging targets. Here, we show that target-optimized detection patterns can yield significantly improved DOT reconstructions in both in silico and experimental tests. Applying reciprocity, we can further iteratively optimize both illumination and detection patterns and show that these simultaneously optimized source/detection patterns outperform predetermined patterns in simulation settings. In addition, we show media-adaptive measurement data compression methods enable wide-field DOT systems to recover highly complex inclusions inside optically-thick media with reduced background artifacts. Furthermore, using truncated optimized patterns shows an improvement of 2-4× in increased speed of data acquisition and reconstruction without significantly losing image quality. The proposed method can be readily extended for additional data dimensions such as spectrum and time.

Integrating terrain structure characteristics into generative adversarial nets for hillshade generation

A hillshade is a visualization technique that represents three-dimensional terrain in a two-dimensional plane by illumination mapping. The digital relief shading promotes the visualization of the terrain efficiently using DEM data. However, compared with manual shading, the digital algorithm-based method still has a gap in the visual effect of illumination strategy and terrain generalization. The typical landform characteristics and micro-geomorphic properties usually are destroyed. The reason is that the complexity of illumination rules is hard to summarize for different terrain phenomena. In this study, namely the data-driven artificial intelligent method, an alternative strategy based on generative adversarial networks (GANs) is proposed rather than the rule-based method. The DEM and the terrain skeleton lines are input to the model and part of the manual relief shading of Swisstopo is used for the training samples. Through the GAN training and learning, the manual skill imbedded in the hillshade is discovered in the generation model. The results show that the proposed model performs better than the digital relief shading on various landforms in aesthetic visualization and geo-scientific representation. Compared to other models, including other convolution neural network (CNN) based methods, terrain structure is maintained more significantly through the proposed model.

Energy efficiency of solar illuminated vertical farms with different illumination strategies

Energy efficiency of a solar illuminated vertical farm is investigated considering different illumination scenarios. The analysis considers the energy consumed by a typical vertical farming facility for heating, cooling and lighting with and without ventilation. Different illumination scenarios consider utilizing fluorescent reflectors converting green light, which is reflected by green leaved produce, to red to increase photosynthetic production, IR filters preventing solar wavelengths, which do not contribute to photosynthetic growth, to enter the facility to reduce cooling load, and light emitting diodes used as a means of complementary/main lighting. Through analyzing 5 different scenarios where these technologies are used in different combinations, the contribution of each technology is identified for 22o, 41o and 60o latitudes in the Northern Hemisphere. It was found that using IR filters is the most effective means of increasing energy efficiency, whereas the impact of using fluorescent coating to energy efficiency is limited in presence of supplementary light emitting diodes illumination. Relying solely on light emitting diodes yield a significant increase in energy consumed to grow per kg of crop. High production rate can be achieved using solar illumination with IR filters, fluorescent coatings and supplementary LED lighting with ventilation that can lead to energy savings of 22 %, 28 %, 29 % with respect to solely relying on LED illumination for 22°N, 41°N, 60°N, respectively. Whereas the corresponding savings, when no ventilation is used are 19 %, 18 %, 13.6 %, for 22°N, 41°N, 60°N, respectively.

Development and Assessment of Multiple Illumination Color Fourier Ptychographic Microscopy for High Throughput Sample Digitization.

In this study, we proposed a multiplexed color illumination strategy to improve the data acquisition efficiency of Fourier ptychography microscopy (FPM). Instead of sequentially lighting up one single channel LED, our method turns on multiple white light LEDs for each image acquisition via a color camera. Thus, each raw image contains multiplexed spectral information. An FPM prototype was developed, which was equipped with a 4×/0.13 NA objective lens to achieve a spatial resolution equivalent to that of a 20×/0.4 NA objective lens. Both two- and four-LED illumination patterns were designed and applied during the experiments. A USAF 1951 resolution target was first imaged under these illumination conditions, based on which MTF curves were generated to assess the corresponding imaging performance. Next, H&E tissue samples and analyzable metaphase chromosome cells were used to evaluate the clinical utility of our strategy. The results show that the single and multiplexed (two- or four-LED) illumination results achieved comparable imaging performance on all the three channels of the MTF curves. Meanwhile, the reconstructed tissue or cell images successfully retain the definition of cell nuclei and cytoplasm and can better preserve the cell edges as compared to the results from the conventional microscopes. This study initially validates the feasibility of multiplexed color illumination for the future development of high-throughput FPM scanning systems.

Unveiling the limits of precision in iterative MINFLUX.

In single-molecule microscopy, a big question is how precisely we can estimate the location of a single molecule. Our research shows that by using iterative localisation microscopy and factoring in the prior information, we can boost precision and reduce the number of photons needed. Leveraging the Van Trees inequality aids in determining the optimal precision achievable. Our approach holds promise for wider application in discerning the optimal precision across diverse imaging scenarios, encompassing various illumination strategies, point spread functions and overarching control methodologies.

High-precision and low-noise dielectric tensor tomography using a micro-electromechanical system mirror.

Dielectric tensor tomography is an imaging technique for mapping three-dimensional distributions of dielectric properties in transparent materials. This work introduces an enhanced illumination strategy employing a micro-electromechanical system mirror to achieve high precision and reduced noise in imaging. This illumination approach allows for precise manipulation of light, significantly improving the accuracy of angle control and minimizing diffraction noise compared to traditional beam steering approaches. Our experiments have successfully reconstructed the dielectric properties of liquid crystal droplets, which are known for their anisotropic structures, while demonstrating a notable reduction in the background noise of the images. Additionally, the technique has been applied to more complex samples, revealing its capability to achieve a high signal-to-noise ratio. This development represents a significant step forward in the field of birefringence imaging, offering a powerful tool for detailed study of materials with anisotropic properties.

Compact three-dimensional fluorescence spectroscopy and its application in food safety

Three-Dimensional fluorescence spectroscopy is an indispensable tool for identification of substances by virtue of its ability to supply abundant characteristic information and high spectral resolution. In this work, we present a compact three-dimensional fluorescence spectrometer utilizing 18 different wavelength LEDs as excitation light source, instead of the conventional bulky multi-wavelength illumination strategy. This spectrometer can in situ obtain the spectral information accurately and fast, while reducing the weight and volume of system. To show its broad biochemical utility, we utilize the compact spectrometer to measure four types of vegetable oil samples from different origins, and analyzed their spectral characteristics. Moreover, we construct a deep learning model based on the 2D-CNN network to detect sesame oil adulteration qualitatively and quantitively. The Compact fluorescence spectrometer proposed has the potential to classify and grade food oils in situ, addressing the problem of sesame oil adulteration on the market.

Enhanced fluorescence blinking of AF647 fluorophores in Mowiol via violet and UV light induced recovery for superior localization microscopy

Blinking of fluorophores is essential in the context of single molecule localization-based optical super-resolution microscopy methods. To make the fluorescence molecule undergo blinking specific complex chemical mounting buffer systems, combined with suitable oxygen scavengers, and reducing agents are required. For instance to realise blinking in widely used fluorescence tags, like Alexa Fluor 647 (AF647), they are to be mounted on anti-fading buffer such as Mowiol and reducing agent such as Beta (β) - ME. However, the quality of the super-resolved images is decided by the total number of blinking events or in other words net duration for which the fluorescence blinking persists. In this paper we investigate how a violet and UV light induced fluorescence recovery mechanism can enhance the duration of fluorescence blinking. Our study uses AF647 dye conjugated with Phalloidin antibody in U87MG cell line mounted on Mowiol and β - ME. On the basis of the investigation we optimize the intensity, at the sample plane, of fluorescence excitation laser at 638 nm and fluorescence recovery beam at 405 nm or in the UV giving the maximum possible fluorescence blinking duration. We observe that the longer blinking duration, using the optimized illumination scheme, has brought down the resolution in the super-resolved image, as given by Fourier Ring Correlation method, from 168 nm to 112 nm, while the separation between two nearby resolvable filaments has been brought down to ≤ 60 nm.

Beam shaping in light-sheet microscopy: an experimental analysis

Abstract Thanks to its unique optical sectioning capability, light-sheet fluorescence microscopy has proven to be a powerful technique for volumetric imaging of entire model organisms with high spatial and temporal resolution. For light sheet generation with scanned laser beams, holographic beam shaping offers precise control over the optical fields exciting the fluorescence. Various illumination schemes have been proposed, aiming for best image quality with regard to axial resolution, optical sectioning, illumination homogeneity and photobleaching while at the same time retaining a large field of view. Here, we have engineered and characterized a variety of beams and analyzed their imaging performance by using phantom samples and zebrafish embryos. These data may assist researchers to select the light sheet best suited to the imaging application at hand.

Complex Spatial Illumination Scheme Optimization of Backscattering Mueller Matrix Polarimetry for Tissue Imaging and Biosensing.

Polarization imaging and sensing techniques have shown great potential for biomedical and clinical applications. As a novel optical biosensing technology, Mueller matrix polarimetry can provide abundant microstructural information of tissue samples. However, polarimetric aberrations, which lead to inaccurate characterization of polarization properties, can be induced by uneven biomedical sample surfaces while measuring Mueller matrices with complex spatial illuminations. In this study, we analyze the detailed features of complex spatial illumination-induced aberrations by measuring the backscattering Mueller matrices of experimental phantom and tissue samples. We obtain the aberrations under different spatial illumination schemes in Mueller matrix imaging. Furthermore, we give the corresponding suggestions for selecting appropriate illumination schemes to extract specific polarization properties, and then provide strategies to alleviate polarimetric aberrations by adjusting the incident and detection angles in Mueller matrix imaging. The optimized scheme gives critical criteria for the spatial illumination scheme selection of non-collinear backscattering Mueller matrix measurements, which can be helpful for the further development of quantitative tissue polarimetric imaging and biosensing.

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells.

Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin - a protein located in the focal adhesion complex - and actin in human osteosarcoma cells.

Line Illumination in Linear Array Photoacoustic Imaging Using a Powell Lens: A Proof-of-Concept Study

Photoacoustic imaging (PAI) is a rapidly developing biomedical imaging technology. Linear array-based photoacoustic tomography (LA-PAT) is one of the most popular configurations of cross-sectional PAI due to its simplicity and clinical translatability. However, when using an optical fiber for LA-PAT, the optical beam shape is deformed due to rapid divergence and, therefore, a larger area on the tissue is illuminated (and the illumination across the linear array is non-uniform), leading to the acquisition of PA signals outside the desired cross-section, which generates artifacts and degrades image resolution. A Powell lens is an optical element that converts a circular beam profile to a nearly linear flat-top profile. In this paper, a Powell lens is used to generate a uniform line illumination scheme that is evaluated with Zemax OpticStudio 2023 R1.02. The system is then characterized experimentally, and the performance is compared with a conventional illumination scheme in LA-PAT.

High-resolution display screen as programmable illumination for Fourier ptychography

We introduce a new programmable illumination strategy for Fourier ptychographic microscopy (FPM), utilizing a high-resolution organic light-emitting diode (OLED) display screen at the vicinity of the sample to achieve super-resolution and quantitative phase imaging of biological samples. High-density pixels in the OLED screen allow for the angle-scanning illumination required for FP by placing the sample slide directly on the screen, where multiple images of the sample are captured while scanning the pixels on the screen. We discuss the design of the OLED-FP illumination and the corresponding image reconstruction strategies and experimentally validate the super-resolution and phase imaging capabilities of FP using a smartphone screen as illumination. Further, we extend our method to patterned illumination strategies, which multiplexes multiple illumination pixels to achieve full-field FP imaging with a reduced number of measurements, where we identify an optimal illumination pattern and density to maximize the imaging speed without sacrificing the image quality. Our approach offers a simple and compact programmable illuminator that can effectively transform any microscope into a compact FPM with resolution improvement and phase imaging capabilities.

High-resolution assessment of multidimensional cellular mechanics using label-free refractive-index traction force microscopy

A critical requirement for studying cell mechanics is three-dimensional assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), which simultaneously quantifies the volumetric morphology and traction force of cells using a high-speed illumination scheme with 0.5-Hz temporal resolution. Without labelling, our method enables quantitative analyses of dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix. When combined with a constrained total variation-based deconvolution algorithm, it provides 0.55-Pa shear and 1.59-Pa normal traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate its utility by assessing the effects of compromised intracellular stress and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.