Confocal Aperture Research Articles

Suppression of natural lens fluorescence in fundus autofluorescence measurements: review of hardware solutions.

Fluorescence lifetime imaging ophthalmoscopy (FLIO), a technique for investigating metabolic changes in the eye ground, can reveal the first signs of diseases related to metabolism. The fluorescence of the natural lens overlies the fundus fluorescence. Although the influence of natural lens fluorescence can be somewhat decreased with mathematical models, excluding this influence during the measurement by using hardware enables more exact estimation of the fundus fluorescence. Here, we analyze four 1-photon excitation hardware solutions to suppress the influence of natural lens fluorescence: aperture stop separation, confocal scanning laser ophthalmoscopy, combined confocal scanning laser ophthalmoscopy and aperture stop separation, and dual point confocal scanning laser ophthalmoscopy. The effect of each principle is demonstrated in examples. The best suppression is provided by the dual point principle, realized with a confocal scanning laser ophthalmoscope. In this case, in addition to the fluorescence of the whole eye, the fluorescence of the anterior part of the eye is detected from a non-excited spot of the fundus. The intensity and time-resolved fluorescence spectral data of the fundus are derived through the subtraction of the simultaneously measured fluorescence of the excited and non-excited spots. Advantages of future 2-photon fluorescence excitation are also discussed. This study provides the first quantitative evaluation of hardware principles to suppress the fluorescence of the natural lens during measurements of fundus autofluorescence.

Optical clearing of living brains with MAGICAL to extend i nvivo imaging.

SummaryTo understand brain functions, it is important to observe directly how multiple neural circuits are performing in living brains. However, due to tissue opaqueness, observable depth and spatiotemporal resolution are severely degraded in vivo. Here, we propose an optical brain clearing method for in vivo fluorescence microscopy, termed MAGICAL (magical additive glycerol improves clear alive luminance). MAGICAL enabled two-photon microscopy to capture vivid images with fast speed, at cortical layer V and hippocampal CA1 in vivo. Moreover, MAGICAL promoted conventional confocal microscopy to visualize finer neuronal structures including synaptic boutons and spines in unprecedented deep regions, without intensive illumination leading to phototoxic effects. Fluorescence emission spectrum transmissive analysis showed that MAGICAL improved in vivo transmittance of shorter wavelength light, which is vulnerable to optical scattering, thus unsuited for in vivo microscopy. These results suggest that MAGICAL would transparentize living brains via scattering reduction.

Ultrastructure and hemodynamics of microaneurysms in retinal vein occlusion examined by an offset pinhole adaptive optics scanning light ophthalmoscope.

Retinal microaneurysms (MAs) associated with retinal vein occlusions often cause macular edema due to vascular leakage from the MAs, which can lead to severe vision loss. However, studies using conventional imaging modalities have not shown a significant association between MAs and retinal functional changes. The recent technological advancements to the adaptive optics scanning light ophthalmoscope (AOSLO) have enabled real-time observation of the human retinal microvasculature. Additionally, offsetting the confocal aperture in the AOSLO enables the blocking of specular reflection from the inner retina and the enhancement of the image contrast of the retinal capillaries. This study investigated the ultrastructure and hemodynamics of MAs examined by structural images and perfusion maps of the offset pinhole AOSLO and evaluated their associations with vascular leakage on fluorescein angiography. Our results show the diverse configurations of the MAs, some of which are occasionally accompanied by a cap structure on the aneurysmal surface. Moreover, the morphological and hemodynamic changes were significantly associated with vascular leakage.

Depth-Profiling Buried Interfaces within Polymer Electrolyte Membranes

Confocal Raman microscopy is being adapted to probe interfaces within bipolar membranes and ionomer-graphene composites of interest for use in fuel cell and related electrochemical devices [1-2]. Although versatile and adaptable to in-situ measurements, confocal Raman microscopy depth-profiling has pitfalls when applied to bulk membranes. Optical aberration and sensitivity to light scattering from sample regions outside the probe beam focus limit depth resolution. With these constraints in mind, initial studies focused on detecting single layer graphene (SLG) as a barrier between two Nafion membranes (Nafion 211, 25 µm thickness). These graphene-sandwich membranes have shown high selectivity for proton transport across electrochemical cells [2]. Using a microscope equipped with oil-immersion optics to provide refractive index matching, the graphene layer was easily detected in confocal Raman microscopy depth-profiles. Spectra were sensitive to defects and stress within the buried graphene layer. Building on the ability to probe membrane-supported SLG, SLG adsorbed to a glass microscope coverslip (SLG/SiO2) was adapted as a platform for confocal aperture alignment and probe volume characterization [1]. Using SLG/SiO2to estimate the limiting axial spatial resolution, the junction separating the AEM/CEM (anion exchange membrane / cation exchange membrane) phases of bipolar polymer membrane (Fumasep FBM) was probed and shown to be consistent with roughness at the boundary on the order of a few micrometers (~3 µm). Continuing efforts are aimed at developing 2-dimensional (x-, y-direction) Raman spatial maps of buried polymer membrane interfaces and model AEM/CEM interfaces for high spatial resolution (< 10 nm) neutron reflectometry depth profiling. 1. Korzeniewski, C.; Kitt, J.P.; Bukola, S.; Creager, S.E.; Minteer, S.D.; Harris, J.M. ”Single layer graphene for estimation of axial spatial resolution in confocal Raman microscopy depth profiling” Anal. Chem.2019, 91, 1049 (DOI:10.1021/acs.analchem.8b04390).2. Bukola, S.; Liang, Y.; Korzeniewski, C.; Harris, J.M.; Creager, S.E. “Selective proton / deuteron transport through Nafion | graphene | Nafion sandwich structures at very high current density” J. Am. Chem. Soc. 2018, 140, 1743. (DOI: 10.1021/jacs.7b10853)

Confocal terahertz SAR imaging of hidden objects through rough-surface scattering.

Terahertz (THz) radar imaging has gained great interests in various applications due to its capability of deep penetration in some specific contents such as plastic and non-conductive materials without water. However, the image quality would be highly degraded by the scattering of the rough surfaces, which remains a challenge in the area. Here, we propose a confocal terahertz synthetic aperture radar (SAR) to alleviate the scattering issues with both improved signal-to-noise ratio (SNR) and resolution. Inspired by the confocal non-line-of-sight imaging in visible wavelength, a convex lens is added into the conventional SAR system to localize the effect of scattering within each spatial sampling point by the confocal configuration. Then, the random phase aberration caused by scattering could be corrected through a simple shift in temporal domain. The performance of the proposed method under different roughness occlusions is evaluated through experiments. All the results demonstrate great enhancements in both resolution and SNR over the conventional methods.

Simple differential digital confocal aperture to improve axial response of line-scanning confocal microendoscopes.

Line-scanning confocal microendoscopy offers video-rate cellular imaging of scattering tissue with relatively simple hardware, but its axial response is inferior to that of point-scanning systems. Based on Fourier optics theory, we designed differential confocal apertures with a simple subtraction technique to improve the line-scanning sectioning performance. Taking advantage of digital slit apertures on a digital light projector and a CMOS rolling shutter, we demonstrate real-time optical sectioning performance comparable to point scanning in a dual-camera microendoscope (<$6,000). We validate the background rejection capability when imaging porcine columnar epithelium stained with fluorescent contrast agents with different uptake mechanisms and staining properties.

Multiplexed holographic non-axial-scanning slit confocal fluorescence microscopy.

A non-axial-scanning multi-plane microscopic system incorporating multiplexed volume holographic gratings and slit array detection to simultaneously acquire optically sectioned images from different depths is presented. The proposed microscopic system is configured such that multiplexed volume holographic gratings are utilized to selectively produce axial focal points in two or more planes inside the sample, and then to use confocal slit apertures to simultaneously image these multiple planes onto corresponding detection areas of a CCD. We describe the design, implementation, and experimental data demonstrating this microscopic system's ability to obtain optically sectioned multi-plane images of fluorescently labeled standard micro-spheres and tissue samples without scanning in axial directions.

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy.

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

Basic principles of confocal microscopy of cаlcium signals

Background: The study of fast processes occurring in living cells, for example, the dynamics of calcium ions or other ions, is actual for modern biophysics, physiology and medicine and can be carried out by laser scanning confocal microscopy. Confocal microscopy as a method of studying biological objects has a number of features requiring an understanding of the delicate mechanisms underlying it. Objectives: The aim of the work is the description of principles and details of measurements of calcium signals in the living cells using laser scanning confocal microscopy and fluorescent indicators. Materials and methods: The analysis of the methodological and practical aspects of studies of calcium signals was carried out by confocal microscopy in the work. Results: Confocal microscopy allows to study the changes in the concentration of free calcium within the cell and even a small part of it by a fluorescent dye, which increases the contrast in comparison with conventional fluorescence microscopy through an additional confocal aperture located in the front of the detector and the use of laser light source and scanning the object. To register calcium signals it is necessary to make a selection of a number of adequate parameters: the registration frequency, the size of a single scanning element (pixel), the sensitivity of the detector, the intensity of laser irradiation, the diameter of the confocal gap, the corresponding filters for exciting and emitting the fluorescent dye used and the corresponding dichroic mirror. An important stage of a setup of a confocal system is the determination of the value of the scattering function of a point. Compensation of the process of bleaching of the fluorescence dye and reduction of phototoxicity, minimization of the scattering process of the image allow to increase a reproducibility of the experiments. Conclusions: Using modern laser scanning confocal microscopes for registration of calcium signals from a small group of channels (for example, rianodine receptors) or the cell part, it is necessary to carefully select the parameters of the hardware setup, which are determined by the nature of the object of study and the peculiarities of the experimental conditions. This will allow to receive the reliable information about the functioning of both individual channels and the mechanisms of Ca2+ signaling of the whole cell or its part.

A portable fiber-optic raman spectrometer concept for evaluation of mineral content within enamel tissue.

BackgroundMeasurement of tooth enamel mineralization using a clinically viable method is essential since variation of mineralization may be used to monitor caries risk or in assessing the effectiveness of remineralization therapy. Fiber optic Raman systems are becoming more affordable and popular in context of biomedical applications. However, the applicability of fiber optic Raman systems for measurement of mineral content within enamel tissue has not been elucidated significantly in the prior literature.Material and MethodsHuman teeth with varying degrees of enamel mineralization were selected. In addition alligator, boar and buffalo teeth which have increasing amount of mineral content, respectively, were also included as another set of samples. Reference Raman measurements of mineralization were performed using a high-fidelity confocal Raman microscope.ResultsAnalysis of human teeth by research grade Raman system indicated a 2-fold difference in the Raman intensities of v1 symmetric-stretch bands of mineral-related phosphate bonds and 7-fold increase in mineral related Raman intensities of animal teeth. However, fiber optic system failed to resolve the differences in the mineralization of human teeth.ConclusionsThese results indicate that the sampling volume of fiber optic systems extends to the underlying dentin and that confocal aperture modification is essential to limit the sampling volume to within the enamel. Further research efforts will focus on putting together portable Raman systems integrated with confocal fiber probe. Key words:Enamel, mineral content, raman spectroscopy.

Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model.

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes determined from the intensity decays. Here, we present a new technique to suppress the autofluorescence of the crystalline lens by introducing an annular stop into the detection light path, which we call Schweitzer's principle. The efficacy of annular stops with an outer diameter of 7 mm and inner diameters of 1 to 5 mm are analyzed in an experimental setup using a model eye based on fluorescent dyes. Compared to the confocal principle, Schweitzer's principle with an inner diameter of 3 mm is able to reduce the simulated crystalline lens fluorescence to 4%, while 42% of the simulated retina fluorescence is preserved. Thus, we recommend the implementation of Schweitzer's principle in scanning laser ophthalmoscopes used for fundus autofluorescence measurements, especially the FLIO device, for improved image quality.

Extrafoveal Cone Packing in Eyes With a History of Retinopathy of Prematurity.

PurposeTo study the density and packing geometry of the extrafoveal cone photoreceptors in eyes with a history of retinopathy of prematurity (ROP). We used a multimodal combination of adaptive optics (AO) scanning light ophthalmoscopy (SLO) and optical coherence tomography (OCT).MethodsCones were identified in subjects (aged 14–26 years) with a history of ROP that was either severe and treated by laser ablation of avascular peripheral retina (TROP; n = 5) or mild and spontaneously resolved, untreated (UROP; n = 5), and in term-born controls (CT; n = 8). The AO-SLO images were obtained at temporal eccentricities 4.5°, 9°, 13.5°, and 18° using both confocal and offset apertures with simultaneous, colocal OCT images. Effects of group, eccentricity, and aperture were evaluated and the modalities compared.ResultsIn the SLO images, cone density was lower and the packing pattern less regular in TROP, relative to CT and UROP retinae. Although SLO image quality appeared lower in TROP, root mean square (RMS) wavefront error did not differ among the groups. In TROP eyes, cone discrimination was easier in offset aperture images. There was no evidence of cone loss in the TROP OCT images.ConclusionsLow cone density in TROP confocal SLO images may have resulted from lower image quality. Since AO correction in these eyes was equivalent to that of the control group, and OCT imaging showed no significant cone loss, the optical properties of the inner retina or properties of the cones themselves are likely altered in a way that affects photoreceptor imaging.

Generation of a spherical focal spot in a 4Pi focusing system through reversing the radiation of infinite biconical antenna

A method of generating spherical focal spot is demonstrated through reversely propagating and tightly focusing the electric field at the pupil plane radiated from an infinite biconical antenna centered at the foci of two confocal high numerical aperture aplanatic objective lenses. The required incident field distribution at the pupil plane is found by solving the inverse problem of radiation from an infinite biconical antenna using the Richards–Wolf vectorial diffraction theory. Numerical simulations show that a focal spot of excellent spherical symmetry along with extremely low sidelobe (~4.72% of the peak) and diffraction limited spot size of 0.432λ can be obtained in a 4Pi focusing system. It is also shown that the generated focal spot can be shifted along the optical axis while still maintaining its spherical shape and diffraction limited size through introducing a phase difference between the pupil illumination fields of the two objective lenses used in the 4Pi focusing system.

Design and Performance of a Multi-Point Scan Confocal Microendoscope.

Confocal fluorescence microendoscopy provides high-resolution cellular-level imaging via a minimally invasive procedure, but requires fast scanning to achieve real-time imaging in vivo. Ideal confocal imaging performance is obtained with a point scanning system, but the scan rates required for in vivo biomedical imaging can be difficult to achieve. By scanning a line of illumination in one direction in conjunction with a stationary confocal slit aperture, very high image acquisition speeds can be achieved, but at the cost of a reduction in image quality. Here, the design, implementation, and experimental verification of a custom multi-point aperture modification to a line-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution of a line-scan system while maintaining high imaging rates. In addition, compared to the line-scanning configuration, previously reported simulations predicted that the multi-point aperture geometry greatly reduces the effects of tissue scatter on image quality. Experimental results confirming this prediction are presented.

Non-common path aberration correction in an adaptive optics scanning ophthalmoscope.

The correction of non-common path aberrations (NCPAs) between the imaging and wavefront sensing channel in a confocal scanning adaptive optics ophthalmoscope is demonstrated. NCPA correction is achieved by maximizing an image sharpness metric while the confocal detection aperture is temporarily removed, effectively minimizing the monochromatic aberrations in the illumination path of the imaging channel. Comparison of NCPA estimated using zonal and modal orthogonal wavefront corrector bases provided wavefronts that differ by ~λ/20 in root-mean-squared (~λ/30 standard deviation). Sequential insertion of a cylindrical lens in the illumination and light collection paths of the imaging channel was used to compare image resolution after changing the wavefront correction to maximize image sharpness and intensity metrics. Finally, the NCPA correction was incorporated into the closed-loop adaptive optics control by biasing the wavefront sensor signals without reducing its bandwidth.

In vivo adaptive optics microvascular imaging in diabetic patients without clinically severe diabetic retinopathy.

We used a confocal adaptive optics scanning laser ophthalmoscope (AOSLO) to image the retina of subjects with non-proliferative diabetic retinopathy (NPDR). To improve visualization of different retinal features, the size and alignment of the confocal aperture were varied. The inner retinal layers contained clearly visualized retinal vessels. In diabetic subjects there was extensive capillary remodeling despite the subjects having only mild or moderate NPDR. Details of the retinal microvasculature were readily imaged with a larger confocal aperture. Hard exudates were observed with the AOSLO in all imaging modes. Photoreceptor layer images showed regions of bright cones and dark areas, corresponding in location to overlying vascular abnormalities and retinal edema. Clinically undetected intraretinal vessel remodeling and varying blood flow patterns were found. Perifoveal capillary diameters were larger in the diabetic subjects (p<0.01), and small arteriolar walls were thickened, based on wall to lumen measurements (p<.05). The results suggest that existing clinical classifications based on lower magnification clinical assessment may not adequately measure key vascular differences among individuals with NPDR.

Retromode imaging: Review and perspectives

Retromode imaging with infrared lasers is a novel imaging method which has been made possible by the newly introduced confocal scanning laser ophthalmoscope. Retromode imaging uses a laterally deviated confocal aperture with a central stop, which creates a shadow and allows deep retinal and retinal pigment epithelium changes to be visualized as pseudo-3-dimensional images. Its clinical value coupled with its simple, rapid, and noninvasive nature is increasingly appreciated. The combination of retromode imaging with conventional imaging methods such as fundus photography, fluorescein angiography, and optical coherence tomography can help to precisely and comprehensively evaluate pathophysiologic features of retinal disorders. This review summarizes basic principles of imaging and retromode findings in various retinal disorders and is expected to guide future investigations of retromode imaging.

Phase‐apodisation technique to extend depth of field for high‐frequency ultrasound imaging

The quality of focusing in high-frequency ultrasound imaging is significantly affected by the length of the depth of field (DOF). A dual-concentric transducer combined with a phase-apodisation scheme is presented to achieve the extended DOF maintaining signal-to-noise ratio. As a preliminary study, computational simulation by using a Field-II program is conducted to demonstrate the feasibility. A dual-concentric transducer is composed of a disc- and a ring-type element with confocal apertures. When two input signals with 0° and 180° phases are simultaneously applied to the inner and outer elements, a bifocal-zone is generated in the axial direction. The overall − 6 dB DOF is 40% longer compared with a single element transducer. Thus, the proposed scheme can be a potential approach to increase the DOF for high-frequency ultrasound imaging.

Imaging of Vascular Wall Fine Structure in the Human Retina Using Adaptive Optics Scanning Laser Ophthalmoscopy

To improve the ability to image the vascular walls in the living human retina using multiply-scattered light imaging with an adaptive optics scanning laser ophthalmoscope (AOSLO). In vivo arteriolar wall imaging was performed on eight healthy subjects using the Indiana AOSLO. Noninvasive imaging of vascular mural cells and wall structure were performed using systematic control of the position of a 10× Airy disk confocal aperture. Retinal arteries and arterioles were divided into four groups based on their lumen diameters (group 1: ≥100 μm; group 2: 50-99 μm; group 3: 10-49 μm; group 4: <10 μm). Fine structure of retinal vasculature and scattering behavior of erythrocytes were clearly visualized in all eight subjects. In group 1 vessels the mural cells were flatter and formed the outer layer of regularly spaced cells of a two (or more) layered vascular wall. In the vessels of groups 2 and 3, mural cells were visualized as distinct cells lying along the lumen of the blood vessel, resulting in a wall of irregular thickness. Vascular wall components were not readily identified in group 4 vessels. Our results show that retinal vascular mural cells and wall structure can be readily resolved in healthy subjects using AOSLO with multiply scattered light imaging for retinal vessels with a lumen diameter greater than or equal to 10 μm. Our noninvasive imaging approach allows direct assessment of the cellular structure of the vascular wall in vivo with potential applications in retinal vascular diseases such as diabetes and hypertension.

In vivo imaging of retinal pigment epithelium cells in age related macular degeneration.

Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgan's method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm(2), which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health.