Annular Illumination Research Articles

Axial peak extraction in the measurement of subsurface structures with annular illumination confocal microscopy

Abstract Three-dimensional dark-field confocal microscopy (DFCM) using annular illumination and complementary detection apertures is capable of detecting subsurface defects in ultra-precision optical components. In this study, a DFCM tuning algorithm for large apertures is proposed based on the sinc2 tuning model. This algorithm provides a reliable and theoretically accurate method for extracting the axial position during the measurement of subsurface defects. The rationality and accuracy of this method are verified through simulations and experimental results.

A Miniaturized Large-Field Fundus Optical System Based on Aspheric Imaging and Non-Coaxial Illumination

Many diseases produce pathological changes in the fundus; analyzing the retinopathy of the fundus could help diagnose diseases in time. A fundus camera is a medical imaging device that specializes in taking fundus images to diagnose hypertension, coronary heart disease, diabetes, and others. The fundus optical imaging system is the core part of it. Nevertheless, the conventional fundus optical imaging system is large and not suitable for mobile examination and follow-up use. So, it has not been widely used in medical institutions. In this paper, a miniaturized fundus optical imaging system based on aspheric technology and non-coaxial illumination is proposed. The length of the imaging system is only 34.6 mm, the field of view is 50°, and the MTF curve is greater than 0.2 at 100 lp/mm, which can resolve the structure of 5 um. The illumination system adopts a non-coaxial annular array illumination structure to avoid occlusion of the imaging system. Our study effectively tackles the pressing predicament of fundus optical system miniaturization. This innovative paradigm harbors the potential to revolutionize fundus image data acquisition, propelling the field of fundus diagnosis forward and efficiently catering to crucial applications, improving the versatility of fundus examination, and providing technical support for the intelligent diagnosis system.

Radially polarized light in single particle optical extinction microscopy identifies silver nanoplates

Quantifying the optical extinction cross section of a single plasmonic nanoparticle (NP) has recently emerged as a powerful method to characterize the NP morphometry, i.e., size and shape, with a precision comparable to electron microscopy while using a simple optical microscope. Here, we enhance the capabilities of extinction microscopy by introducing a high numerical aperture annular illumination coupled with a radial polarizer to generate a strong axial polarization component. This enables us to probe the NP response to axial polarized light, and, in turn, to distinguish flat-lying nanoplates from other geometries. Polarization-resolved optical extinction cross sections were acquired on 219 individual colloidal silver NPs of a nominally triangular nanoplate shape but, in practice, exhibiting heterogeneous morphometries, including decahedrons and non-plate spheroids. An unsupervised machine learning cluster analysis algorithm was developed, which allowed us to separate NPs into different groups, owing to the measured differences in cross sections. Comparison of the measurements with a computational model of the absorption and scattering cross section accounting for nanoplates of varying geometries beyond simple triangles provided insight into the NP shape of each group. The results provide a significant improvement of polarization-resolved optical extinction microscopy to reconstruct NP shapes, further boosting the utility of the method as an alternative to electron microscopy analysis.

High‐Speed Multi‐Modal Extended Depth‐of‐Field Microscopy with an Electrically Tunable Lens

AbstractHigh spatial resolution imaging over the entire volume for intravital imaging of biological specimens has long been vital. However, the depth‐of‐field (DOF) and spatial resolution are intrinsically interdependent. Here, a new extended DOF (EDOF) microscopy technique based on partially coherent annular illumination is proposed, termed AI‐EDOF, by combining an electrically tunable lens with a conventional microscope with modified illumination, for motion‐free, high spatial resolution real‐time all‐in‐focus imaging over thick volumes. EDOF imaging is obtained with spatial resolution up to near the incoherent diffraction limit (388 nm, 20/ 0.8NA) and temporal resolution 30 fps. Moreover, it is demonstrated that the coupled phase and absorption components of the complex refractive index in optical diffraction tomography microscopy can be solved by focus scanning. The Richardson‐Lucy deconvolution with total variation regularization is adopted for deblurring and suppressing noise under low light efficiency high‐speed exposure. To demonstrate the capabilities of the proposed method, experiments are conducted using fixed transgenic zebrafish larvae, Drosophila larvae and dynamic Caenorhabditis elegans under both transmissive and fluorescent imaging modalities, revealing the proposed approach is prospective to be adopted by broad applications such as pharmacokinetics and tumor immunology.

Image Contrast Enhancement by Using LED Annular Oblique Illumination in Bright-Field Microscopy

Köhler illumination is the most used method for evenly illuminating a sample in bright-field microscopy. However, Köhler illumination cannot provide optimum contrast and high resolution simultaneously. The image contrast for Köhler illumination can be enhanced at the expense of lateral resolution. In this paper, we discuss the main causes of contrast loss with Köhler illumination by simulating unsymmetrical interference, incoherent superposition, and modulation transfer function. We present a method to increase the image contrast by combining the annular oblique illumination with normalization and blind deconvolution. With the progress of optoelectronic devices, the greater advantages of annular oblique illumination will become apparent. The experimental results achieved by using LED annular oblique illumination prove the feasibility of the proposed method.

Parallel synthetic aperture transport-of-intensity diffraction tomography with annular illumination.

Transport-of-intensity diffraction tomography (TIDT) is a recently developed label-free computational microscopy technique that retrieves high-resolution three-dimensional (3D) refractive index (RI) distribution of biological specimens from 3D intensity-only measurements. However, the non-interferometric synthetic aperture in TIDT is generally achieved sequentially through the acquisition of a large number of through-focus intensity stacks captured at different illumination angles, resulting in a very cumbersome and redundant data acquisition process. To this end, we present a parallel implementation of a synthetic aperture in TIDT (PSA-TIDT) with annular illumination. We found that the matched annular illumination provides a mirror-symmetric 3D optical transfer function, indicating the analyticity in the upper half-plane of the complex phase function, which allows for recovery of the 3D RI from a single intensity stack. We experimentally validated PSA-TIDT by conducting high-resolution tomographic imaging of various unlabeled biological samples, including human breast cancer cell lines (MCF-7), human hepatocyte carcinoma cell lines (HepG2), Henrietta Lacks (HeLa) cells, and red blood cells (RBCs).

Adaptive optical quantitative phase imaging based on annular illumination Fourier ptychographic microscopy

Quantitative phase imaging (QPI) has emerged as a valuable tool for biomedical research thanks to its unique capabilities for quantifying optical thickness variation of living cells and tissues. Among many QPI methods, Fourier ptychographic microscopy (FPM) allows long-term label-free observation and quantitative analysis of large cell populations without compromising spatial and temporal resolution. However, high spatio-temporal resolution imaging over a long-time scale (from hours to days) remains a critical challenge: optically inhomogeneous structure of biological specimens as well as mechanical perturbations and thermal fluctuations of the microscope body all result in time-varying aberration and focus drifts, significantly degrading the imaging performance for long-term study. Moreover, the aberrations are sample- and environment-dependent, and cannot be compensated by a fixed optical design, thus necessitating rapid dynamic correction in the imaging process. Here, we report an adaptive optical QPI method based on annular illumination FPM. In this method, the annular matched illumination configuration (i.e., the illumination numerical aperture (NA) strictly equals to the objective NA), which is the key for recovering low-frequency phase information, is further utilized for the accurate imaging aberration characterization. By using only 6 low-resolution images captured with 6 different illumination angles matching the NA of a 10x, 0.4 NA objective, we recover high-resolution quantitative phase images (synthetic NA of 0.8) and characterize the aberrations in real time, restoring the optimum resolution of the system adaptively. Applying our method to live-cell imaging, we achieve diffraction-limited performance (full-pitch resolution of 655,nm at a wavelength of 525,nm) across a wide field of view (1.77,mm^2) over an extended period of time.

Photoacoustic testing of shear viscoelastic properties of soft tissues using annular beam illumination.

This Letter reports a new, to the best of our knowledge, photoacoustic excitation method for evaluating the shear viscoelastic properties of soft tissues. By illuminating the target surface with an annular pulsed laser beam, circularly converging surface acoustic waves (SAWs) are generated, focused, and detected at the center of the annular beam. The shear elasticity and shear viscosity of the target are extracted from the dispersive phase velocity of the SAWs based on the Kelvin-Voigt model and nonlinear regression fitting. Agar phantoms with different concentrations, and animal liver and fat tissue samples have successfully been characterized. Different from previous methods, the self-focusing of the converging SAWs allows sufficient SNR to be obtained even with low pulsed laser energy density, which makes this approach well compatible with soft tissues under both ex vivo and in vivo testing conditions.

Single-path single-shot phase-shifting quantitative phase microscopy with annular bright-field illumination

We propose single-path single-shot phase-shifting quantitative phase microscopy (SSP-QPM) with annular illumination, oil top lens, and wide-field objectives. We analyze the difference between bright-field SSP-QPM, phase-contrast SSP-QPM, and the proposed microscopy by resolution, contrast, and noise. The proposed microscopy enhances the lateral resolution by promoting the numerical-aperture of illumination, improves the image contrast by suppressing multiple-scattering, and reduces dynamic noise by decreasing exposure time. Experiments validate the characters of the proposed microscopy by quantitative phase imaging of oocyte cells, intestinal tissues, and polystyrene microspheres. Besides, we demonstrate the simultaneous recording of fluorescence and quantitative phase images.

Annular illumination in 2D quantitative phase imaging: a systematic evaluation.

Quantitative phase imaging (QPI) is an invaluable microscopic technology for definitively imaging phase objects such as biological cells and optical fibers. Traditionally, the condenser lens in QPI produces disk illumination of the object. However, it has been realized by numerous investigators that annular illumination can produce higher-resolution images. Although this performance improvement is impressive and well documented, the evidence presented has invariably been qualitative in nature. Recently, a theoretical basis for annular illumination was presented by Bao et al. [Appl. Opt.58, 137 (2019)APOPAI0003-693510.1364/AO.58.000137]. In our current work, systematic experimental QPI measurements are made with a reference phase mask to rigorously document the performance of annular illumination. In both theory and experiment, three spatial-frequency regions are identified: low, mid, and high. The low spatial-frequency region response is very similar for disk and annular illumination, both theoretically and experimentally. Theoretically, the high spatial-frequency region response is predicted to be much better for the annular illumination compared to the disk illumination--and is experimentally confirmed. In addition, the mid-spatial-frequency region response is theoretically predicted to be less for annular illumination than for disk illumination. This theoretical degradation of the mid-spatial-frequency region is only slightly experimentally observed. This bonus, although not well understood, further elevates the performance of annular illumination over disk illumination.

Compact dark-field confocal microscopy based on an annular beam with orbital angular momentum.

This study proposes a dark-field confocal microscopy (DFCM) based on fiber-mode excitation-assisted orbital angular momentum annular beam generation with a two-mode fiber to avoid diffraction distortion. The application of optical fibers compresses the DFCM volume, providing new ideas for system miniaturization. System adjustment difficulty is reduced and feasibility is verified by imaging 2D and 3D samples. High imaging contrast is achieved by fully blocking the reflected light and annular illumination with a steady central dark-spot within a propagation distance of 3 m. The application of our scheme can be further extended to detect subsurface defects in optical components and high-contrast biological imaging.

A Novel, Smartphone-Based, Teleophthalmology-Enabled, Widefield Fundus Imaging Device With an Autocapture Algorithm.

PurposeWidefield imaging can detect signs of retinal pathology extending beyond the posterior pole and is currently moving to the forefront of posterior segment imaging. We report a novel, smartphone-based, telemedicine-enabled, mydriatic, widefield retinal imaging device with autofocus and autocapture capabilities to be used by non-specialist operators.MethodsThe Remidio Vistaro uses an annular illumination design without cross-polarizers to eliminate Purkinje reflexes. The measured resolution using the US Air Force target test was 64 line pairs (lp)/mm in the center, 57 lp/mm in the middle, and 45 lp/mm in the periphery of a single-shot retinal image. An autocapture algorithm was developed to capture images automatically upon reaching the correct working distance. The field of view (FOV) was validated using both model and real eyes. A pilot study was conducted to objectively assess image quality. The FOVs of montaged images from the Vistaro were compared with regulatory-approved widefield and ultra-widefield devices.ResultsThe FOV of the Vistaro was found to be approximately 65° in one shot. Automatic image capture was achieved in 80% of patient examinations within an average of 10 to 15 seconds. Consensus grading of image quality among three graders showed that 91.6% of the images were clinically useful. A two-field montage on the Vistaro was shown to exceed the cumulative FOV of a seven-field Early Treatment Diabetic Retinopathy Study image.ConclusionsA novel, smartphone-based, portable, mydriatic, widefield imaging device can view the retina beyond the posterior pole with a FOV of 65° in one shot.Translational RelevanceSmartphone-based widefield imaging can be widely used to screen for retinal pathologies beyond the posterior pole.

In-vivo sub-diffraction adaptive optics imaging of photoreceptors in the human eye with annular pupil illumination and sub-Airy detection.

Adaptive optics scanning light ophthalmoscopy (AOSLO) allows non-invasive visualization of the living human eye at the microscopic scale; but even with correction of the ocular wavefront aberrations over a large pupil, the smallest cells in the photoreceptor mosaic cannot always be resolved. Here, we synergistically combine annular pupil illumination with sub-Airy disk confocal detection to demonstrate a 33% improvement in transverse resolution (from 2.36 to 1.58 μm) and a 13% axial resolution enhancement (from 37 to 32 μm), an important step towards the study of the complete photoreceptor mosaic in heath and disease. Interestingly, annular pupil illumination also enhanced the visualization of the photoreceptor mosaic in non-confocal detection schemes such as split detection AOSLO, providing a strategy for enhanced multimodal imaging of the cone and rod photoreceptor mosaic.

Zone plate-based full-field transmission X-ray microscopy beamline design at nearly diffraction-limited synchrotron radiation facility

With the development of full-field transmission X-ray microscopy (TXM) for research in biology, life science, material science and physics, zone plate-based full-field TXM is becoming a powerful tool for obtaining quantitative internal structural and chemical information at the nanoscale when combined with spectroscopic imaging. For the TXM beamline design, the best choice is to match the phase space needed by the zone plate from the source point. However, for the nearly diffraction-limited synchrotron radiation sources, it is not easy to fully match the needed phase space due to its low-emittance. Using redshift radiation from an undulator, under consideration of experimental requirements and the heat load effect, this paper describes the design of TXM beamline at the nearly diffraction-limited High Energy Photon Source (HEPS). In this design, not only sufficient illumination angle can be acquired, but also a nearly annular illumination can be obtained to match the illumination required for zone plate imaging. Furthermore, about two times photon flux can be obtained at source point and the photons can be fully used in this design using redshift undulator radiation. The presented design can also be informative for the same kind of beamline design at other nearly diffraction-limited synchrotron photon sources.

Fundus image quality assessment for an eye model with cataract using adapted illumination patterns

PurposeIn recent years, different methods have been developed to improve fundus image quality in eyes with cataract. For stray light reduction, we have introduced a segmented LED light source. To analyze the effect of different illumination patterns at different cataract characteristics, we developed an optical cataract eye model. Accordingly, we performed fundus imaging using a fundus camera equipped with a LED light source controlled by a Raspberry Pi.MethodsA customized 24‐fold segmented LED light source was connected to a fundus camera. The eye model consists of a plano‐convex lens, a cover glass, and a test chart with five black‐white test fields. The pupil diameter was set to 7 mm. To test the influence of variable illumination patterns, four different cortical cataract patterns were produced by sand blasting the cover glasses. Three spoke‐like (1Spoke, 4S, 7S) patterns and one crescent‐shaped/CS pattern were realized. To analyze the effect of adapted illumination conditions, the gray‐level co‐occurrence matrix (GLCM) contrasts were calculated. At that, one image was taken at standard annular illumination (all LEDs on) as baseline followed by seven defined adapted configurations, for each type of cataract.ResultsFor the four types of cataracts, the baseline GLCM contrast values were 0.21 (1S), 0.18 (4S), 0.17 (7S), and 0.22 (CS). In each cataract type, the best‐fitting illumination pattern resulted in 0.23 (1S), 0.29 (4S), 0.22 (7S), and 0.23 (CS). Compared to the baseline values, the increases of the GLCM contrasts were 9.5% (1S), 61% (4S), 29% (7S), and 4.5% (CS).ConclusionsWe successfully performed structured LED illumination for different cortical cataract types using an optical eye model. An improvement of fundus image quality could be determined in all investigated cases using the gray‐level co‐occurrence matrix contrast.

Optical design of LED high mast lamp based on TIR freeform lens for annular lighting

The traditional square spot of street lamp is not suitable for the lighting requirements of the annular road lighting. In this paper, a lens design method for producing annular spot of high mast lamp is proposed. The method mainly contains three optical surfaces, in which the inner surface collimates the energy of the centre part of the light source, the total internal reflection surface collimates the light with large angle and low energy and the outer surface refracts all the collimated light to realise the annular illumination distribution on the target surface. In this design, we can change the diameter of the lighting spot through the variation of the inclination angle of the outer surface from 14° to 19° and the diameter of the spot changes from 10.2 m to 12.3 m at the distance of 30 m. The lighting requirements of high mast lighting are realised based on the TIR lens array, in which the average illuminance is more than 20 lx, the illuminance uniformity is more than 0.7 and the light optical utilisation factor is more than 90%.

Resolution-enhanced intensity diffraction tomography in high numerical aperture label-free microscopy

We propose label-free and motion-free resolution-enhanced intensity diffraction tomography (reIDT) recovering the 3D complex refractive index distribution of an object. By combining an annular illumination strategy with a high numerical aperture (NA) condenser, we achieve near-diffraction-limited lateral resolution of 346 nm and axial resolution of 1.2 μm over 130 μ m × 130 μ m × 8 μ m volume. Our annular pattern matches the system’s maximum NA to reduce the data requirement to 48 intensity frames. The reIDT system is directly built on a standard commercial microscope with a simple LED array source and condenser lens adds-on, and promises broad applications for natural biological imaging with minimal hardware modifications. To test the capabilities of our technique, we present the 3D complex refractive index reconstructions on an absorptive USAF resolution target and Henrietta Lacks (HeLa) and HT29 human cancer cells. Our work provides an important step in intensity-based diffraction tomography toward high-resolution imaging applications.

Single-shot wavelength-selective quantitative phase microscopy by partial aperture imaging and polarization-phase-division multiplexing.

We propose a single-shot wavelength-selective quantitative phase microscopy by annular white-light illumination, polarization-phase-division, and parallel phase-shifting. Compared to conventional multi-wavelength incoherence digital holography, the proposed microscopy presents the following merits: no switching of illumination or mechanical scanning, high spatiotemporal phase sensitivity, and single-shot reconstruction at each wavelength. Experiments validate these characteristics by quantitative phase imaging of gratings, cells, and tissues.

Two-point resolution in bright background under partially coherent light

Two-point resolution for ordinary microscopy was studied in a bright background under partially coherent light. The basic equation was derived from the unified theory of image intensity for two point-like objects for bright and dark backgrounds. For an extended Rayleigh’s criterion, it is shown that the two-point resolution in a bright background is noticeably different from that in a dark background. The resolution for annular illumination is also dealt with, and its characteristics are elucidated.

Numerical study on the tight focusing of radially polarized beams with polarization-insensitive metalenses

Radially polarized beam (RPB) could be focused to a tighter spot than a spatially homogeneous polarized beam with a high numerical aperture (NA) objective lens. However, the traditional objective lens suffers from high cost and bulky size. In this paper, polarization-insensitive nanopillar metalenses were designed to execute tight focusing without the requirement of the incident beams to be specific polarized states. It was found that the focal spot of RPB was potentially smaller than that of a linear polarized beam because of the longitudinal component. Diffraction-limited focusing of RPB was achieved by a high NA polarization-insensitive metalens through an annular aperture illumination. This study may contribute to high-resolution photonics and optoelectronic applications.