Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

We have developed a CCD-camera-based nonmydriatic instrument that detects fluorescence from retinal lipofuscin chromophores ("autofluorescence") as a means to indirectly quantify and spatially image the distribution of macular pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing MP, since MP has a competing absorption in the blue-green wavelength region. Projecting a large diameter, 488 nm excitation spot onto the retina, centered on the fovea, but extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP. Spectrally selective detection of the lipofuscin fluorescence reveals an important wavelength dependence of the obtainable image contrast and deduced MP optical density levels, showing that it is important to block out interfering fluorescence contributions in the detection setup originating from ocular media such as the lens. Measuring 70 healthy human volunteer subjects with no ocular pathologies, we find widely varying spatial extent of MP, distinctly differing distribution patterns of MP, and strongly differing absolute MP levels among individuals. Our population study suggests that MP imaging based on lipofuscin fluorescence is useful as a relatively simple, objective, and quantitative noninvasive optical technique suitable to rapidly screen MP levels and distributions in healthy humans with undilated pupils.     

Black Phosphorus/Platinum Heterostructure: A Highly Efficient Photocatalyst for Solar‐Driven Chemical Reactions

A 2D black phosphorus/platinum heterostructure (Pt/BP) is developed as a highly efficient photocatalyst for solar‐driven chemical reactions. The heterostructure, synthesized by depositing BP nanosheets with ultrasmall (≈1.1 nm) Pt nanoparticles, shows strong Pt–P interactions and excellent stability. The Pt/BP heterostructure exhibits obvious P‐type semiconducting characteristics and efficient absorption of solar energy extending into the infrared region. Furthermore, during light illumination, accelerated charge separation is observed from Pt/BP as manifested by the ultrafast electron migration (0.11 ps) detected by a femtosecond pump‐probe microscopic optical system as well as efficient electron accumulation on Pt revealed by in situ X‐ray photoelectron spectroscopy. These unique properties result in remarkable performance of Pt/BP in typical hydrogenation and oxidation reactions under simulated solar light illumination, and its efficiency is much higher than that of other common Pt catalysts and even much superior to that of conventional thermal catalysis. The 2D Pt/BP heterostructure has enormous potential in photochemical reactions involving solar light and the results of this study provide insights into the design of next‐generation high‐efficiency photocatalysts.     

Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination

We present a simple and cost-effective wide-field, depth-sectioning, fluorescence microscope utilizing a commercial multimedia projector to generate excitation patterns on the sample. Highly resolved optical sections of fluorescent pollen grains at 1.9 microm axial resolution are constructed using the structured illumination technique. This requires grid excitation patterns to be scanned across the sample, which is straightforwardly implemented by creating slideshows of gratings at different phases, projecting them onto the sample, and synchronizing camera acquisition with slide transition. In addition to rapid dynamic pattern generation, the projector provides high illumination power and spectral excitation selectivity. We exploit these properties by imaging mouse neural cells in cultures multistained with Alexa 488 and Cy3. The spectral and structural neural information is effectively resolved in three dimensions. The flexibility and commercial availability of this light source is envisioned to open multidimensional imaging to a broader user base.     

Optimization of a three-dimensional digital image correlation system for deformation measurements in extreme environments

An optimized 3D digital image correlation (3D-DIC) system using active optical imaging is developed for accurate shape and 3D deformation measurements in nonlaboratory conditions or extreme high-temperature environments. In contrast to a conventional 3D-DIC system using white or natural light illumination, the proposed active imaging 3D-DIC system is based on a combination of monochromatic lighting and bandpass filter imaging. Because the bandpass filter attached before the imaging lenses allows only the actively illuminated monochromatic light to pass through and blocks all light outside of its bandpass range, the active imaging 3D-DIC system is therefore insensitive to serious variations in ambient light in nonlaboratory environments and to the thermal radiation of hot objects in extreme high-temperature environments. Two challenging experiments that cannot be performed by a conventional 3D-DIC system were carried out to verify the robustness and accuracy of the developed active imaging 3D-DIC system. Because a much wider application range can be achieved with relatively simple and easy-to-implement improvements, the proposed active imaging 3D-DIC system is highly recommended for practical use instead of the conventional 3D-DIC system.     

相移掩模和离轴照明在ArF浸没式光刻中对工艺窗口的影响

研究了交替型相移掩模及离轴照明对65nm分辨率ArF浸没式光刻的影响,在3／4环形照明和3／4四极照明方式下,分别选用传统掩模和交替型相移掩模,研究65nm线宽的密集线条、半密集线条、孤立线条在较大的曝光系统参数范围内,对光刻工艺窗口的改善。并对在不同的照明方式、掩模结构下获得的工艺窗口进行了比较,结果表明：①在较大焦深(DOF)范围内,满足光刻性能要求可以有较大范围的曝光系统参数配置;②相时于传统照明和传统掩模,采用交替型相移掩模或者离轴照明,焦深均可提高100％-150％。     

Ocular aberrations with ray tracing and Shack-Hartmann wave-front sensors: does polarization play a role?

Ocular aberrations were measured in 71 eyes by using two reflectometric aberrometers, employing laser ray tracing (LRT) (60 eyes) and a Shack-Hartmann wave-front sensor (S-H) (11 eyes). In both techniques a point source is imaged on the retina (through different pupil positions in the LRT or a single position in the S-H). The aberrations are estimated by measuring the deviations of the retinal spot from the reference as the pupil is sampled (in LRT) or the deviations of a wave front as it emerges from the eye by means of a lenslet array (in the S-H). In this paper we studied the effect of different polarization configurations in the aberration measurements, including linearly polarized light and circularly polarized light in the illuminating channel and sampling light in the crossed or parallel orientations. In addition, completely depolarized light in the imaging channel was obtained from retinal lipofuscin autofluorescence. The intensity distribution of the retinal spots as a function of entry (for LRT) or exit pupil (for S-H) depends on the polarization configuration. These intensity patterns show bright corners and a dark area at the pupil center for crossed polarization, an approximately Gaussian distribution for parallel polarization and a homogeneous distribution for the autofluorescence case. However, the measured aberrations are independent of the polarization states. These results indicate that the differences in retardation across the pupil imposed by corneal birefringence do not produce significant phase delays compared with those produced by aberrations, at least within the accuracy of these techniques. In addition, differences in the recorded aerial images due to changes in polarization do not affect the aberration measurements in these reflectometric aberrometers.     

Doppler encoded excitation pattern tomographic optical microscopy

Most far-field optical imaging systems rely on lenses and spatially resolved detection to probe distinct locations on the object. We describe and demonstrate a high-speed wide-field approach to imaging that instead measures the complex spatial Fourier transform of the object by detecting its spatially integrated response to dynamic acousto-optically synthesized structured illumination. Tomographic filtered backprojection is applied to reconstruct the object in two or three dimensions. This technique decouples depth of field and working distance from resolution, in contrast to conventional imaging, and can be used to image biological and synthetic structures in fluoresced or scattered light employing coherent or broadband illumination. We discuss the electronically programmable transfer function of the optical system and its implications for imaging dynamic processes. We also explore wide-field fluorescence imaging in scattering media by coherence gating. Finally, we present two-dimensional high-resolution tomographic image reconstructions in both scattered and fluoresced light demonstrating a thousandfold improvement in the depth of field compared to conventional lens-based microscopy.     

Nanophotonic crescent moon structures with sharp edge for ultrasensitive biomolecular detection by local electromagnetic field enhancement effect

We present novel gold nanophotonic crescent moon structures with a sub-10 nm sharp edge, which can enhance local electromagnetic field at the edge area. The formation of unconventional nanophotonic crescent moon structure is accomplished by using a sacrificial nanosphere template and conventional thin film deposition method, which allows an effective batch nanofabrication and precise controls of nanostructure shapes. Unique multiple scattering peaks are observed in a single gold nanocrescent moon with dark-field white light illumination. A 785 nm near-infrared (NIR) diode laser was used as the excitation source to induce the amplified scattering field on the sharp edge of the single gold nanocrescent moon. The Raman scattering spectrum of Rhodamine 6G molecules adsorbed on the single gold nanocrescent moon are characterized, and the Raman enhancement factor of single gold nanocrescent moon is estimated larger than 10(10), which suggests the potential applications of gold nanocrescent moons in ultrasensitive biomolecular detection and cellular imaging using surface enhanced Raman spectroscopy.     

Theoretical development and experimental evaluation of imaging models for differential-interference-contrast microscopy.

Imaging models for differential-interference-contrast (DIC) microscopy are presented. Two- and three-dimensional models for DIC imaging under partially coherent illumination were derived and tested by using phantom specimens viewed with several conventional DIC microscopes and quasi-monochromatic light. DIC images recorded with a CCD camera were compared with model predictions that were generated by using theoretical point-spread functions, computer-generated phantoms, and estimated imaging parameters such as bias and shear. Results show quantitative and qualitative agreement between model and data for several imaging conditions.     

THz Characterization of All-Trans and 9-cis Retinal, Experiment, and Modeling

Two conformations of the retinal molecule have been studied in order to characterize the molecule's THz transmission spectra in both the ground and metastable states. When subjected to an adequate external excitation the retinal molecule can experience a change in conformation and associated THz transmission spectra. In an attempt to accomplish this characterization, the FTIR system was modified to include a simple off axis excitation source inside of the systems sample chamber. Measurements were made of the retinal molecule's THz spectra both with and without external excitation of the molecule. The results gathered were then compared with the results obtained from simulation. Data obtained from two retinal isomers reveal more spectral features at frequencies ~11 - 15 cm^-1 than were predicted for these conformations. The most likely explanation for this is that the material is actually a mixture of several metastable conformations. There is correlation between simulated and measured THz spectra in the ground state at a frequency of 14 cm^-1 for all-trans retinal. The strongest vibrational mode frequency predicted for the 9-cis conformation through modeling was 22 cm^-1, which correlates quite well with the experimental line at 21.3 cm^-1 in the ground state. When the 9-cis samples were exposed to UV illumination there was a noticeable change in the absorption spectra and this line at 21.3 cm^-1 almost disappeared which can be related to the transformation of a 9-cis into a more stable all-trans retinal. The absorption spectra of all-trans retinal that is the most stable conformation showed very weak features in experimental spectra, with some of them changing under illumination.     

Metal nanoparticle plasmon-enhanced light-harvesting in a photosystem I thin film

Silver metal nanoparticle (NP) enhanced fluorescence is investigated in thin films of cyanobacterial Photosystem I trimer complexes (PSI) by correlating confocal laser scanning microscopy, dark-field imaging, and fluorescence lifetime measurements. PSI represents an interesting light-harvesting complex with a 20 nm diameter that is not uniformly contained within the surface-localized plasmon field of the NPs. With weak far-field illumination, 5- to 20-fold fluorescence enhancement is observed for PSI complexes adjacent to NPs, arising from efficient nanoparticle light collection and subsequent localized, surface plasmon excitation of PSI. Enhanced PSI fluorescence is detected most prominently near "rafts" of aggregated NPs that more completely fill the confocal field of view. These results demonstrate opportunities to probe energy transfer within photosynthetic complexes using plasmonic excitation and to design nanostructures for optimizing artificial light-harvesting systems.     

High-throughput flow cytometric DNA fragment sizing

The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel imaging of the fluorescence from individual DNA molecules, stained with a fluorescent intercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement speed and the sample throughput compared to conventional SMFC. Fluorescence bursts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relationship between the number of detected photons per burst and the DNA fragment size was confirmed. Application of this parallel fluorescence imaging method will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.     

Influence of solvent composition on the performance of carbodiimide cross-linked gelatin carriers for retinal sheet delivery

Gelatin is a protein molecule that displays bioaffinity and provides a template to guide retinal pigment epithelial (RPE) cell organization and growth. We have recently demonstrated that the carbodiimide cross-linked gelatin membranes can be used as retinal sheet carriers. The purpose of this work was to further determine the role of solvent composition in the tissue delivery performance of chemically modified biopolymer matrices. The gelatin molecules were treated with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in the presence of binary ethanol/water mixtures with varying ethanol concentrations (70–95 vol%) to obtain the carriers with different cross-linking efficiencies and mechanical properties. Results of melting point measurements and in vitro degradation tests showed that when the cross-linking index reached a high level of around 45 %, the EDC cross-linked gelatin materials have sufficient thermal stability and resistance to enzymatic degradation, indicating their suitability for the development of carriers for retinal sheet delivery. Irrespective of the solvent composition, the chemically modified gelatin samples are compatible toward human RPE cells without causing toxicity and inflammation. In particular, the membrane carriers prepared by the cross-linking in the presence of solvent mixtures containing 80–90 vol% of ethanol have no impact on the proliferative capacity of ARPE-19 cultures and possess good efficiency in transferring and encapsulating the retinal tissues. It is concluded that, except for cell viability and pro-inflammatory cytokine expression, the retinal sheet delivery performance strongly depends on the solvent composition for EDC cross-linking of gelatin molecules.     

Broadband light profile microscopy: a rapid and direct method for thin film depth imaging

Light profile microscopy (LPM) is a recently developed technique of optical inspection that is used to record micrometer scale images of thin film cross-sections on a direct basis. This technique uses a novel right-angle imaging geometry that shows outstanding contrast for subtle interface structures and morphologies that are invisible to conventional methods of inspection. When laser sources are used for sample illumination, image contrast is provided by luminescence and elastic and/or inelastic scatter. When a white-light excitation source is used for LPM, primary contrast is obtained from elastic scatter, while secondary contrast results from refraction, secondary transmission, and secondary reflection from material phases. We term this mode of inspection broadband light profile microscopy (BB-LPM). It is implemented with a compact, easily aligned apparatus and minimal sample preparation, and it shows outstanding interface contrast similar to laser LPM. In this work we demonstrate BB-LPM as a method for direct imaging of the layers structures of a variety of thin film samples of industrial and manufacturing interest.     

量子点荧光材料在照明和显示领域的研究进展

由于量子点具有发光波长尺寸可调谐、发光峰窄、发光效率高和热稳定性等特点,近年来作为一种新型发光材料受到了越来越多的关注。它们不仅可以在电激发下实现自主发光,还可以在光激发下实现光转换,因此在照明和显示领域具有很广阔的应用前景。总结了用量子点荧光材料制备白光发光二极管的6种方法及其研究进展,还介绍了它们在液晶显示领域的应用,最后讨论了目前量子点荧光粉存在的问题和今后的研究方向。     

Resolution enhancement in standing-wave total internal reflection microscopy: a point-spread-function engineering approach.

The theoretical basis for resolution enhancement in standing-wave total internal reflection microscopy (SW-TIRM) is examined. This technique relies on the formation of an excitation field containing super-diffraction-limited spatial-frequency components. Although the fluorescence generated at the object planes contains high-frequency information of the object distribution, this information is lost at the image plane, where the detection optics acts as a low-pass filter. From the perspective of point-spread-function (PSF) engineering, one can show that if this excitation field is translatable experimentally, the high-frequency information can be extracted from a set of images where the excitation fields have different displacement vectors. We have developed algorithms to combine this image set to generate a composite image with an effective PSF that is equal to the product of the excitation field and the Fraunhofer PSF. This approach can easily be extended to incorporate nonlinear excitation modalities into SW-TIRM for further resolution improvement. We theoretically examine high-resolution imaging based on the addition of two-photon, pump-probe, and stimulated-emission depletion methods to SW-TIRM and show that resolution better than 1/20 of the emission wavelength may be achievable.     

Up-converted ultraviolet luminescence of Er<Superscript>3+</Superscript>:BaGd<Subscript>2</Subscript>ZnO<Subscript>5</Subscript> phosphors for healthy illumination

Moderate level of exposure to the solar irradiation containing UV component is essential for health care. To incorporate the UV-emitting phosphors into the commercial YAG-based white light-emitting diode introduces the possibilities of healthy illumination to individuals’ daily lives. 1 mol.% Er³⁺-doped BaGd₂ZnO₅ (BGZ) particles were synthesized via sol-gel method and efficient up-converted luminescence peaked at 380 nm was detected under 480 nm excitation. The mixed phosphors with varied mass ratio of Er³⁺:BGZ and Ce³⁺:YAG particles were encapsulated to form LEDs. The study of the LEDs indicated that the introduction of BGZ component favored the enhancement of color-rendering index and the neutralization of the white light emitting. The WLED with the BGZ/YAG ratio of 8:2 was recommendable for its excellent overall white light luminous performances and UV intensity of 84.55 mW/cm². The UV illumination dose of the WLEDs with mixed YAG and BGZ was controllable by adjusting the ratio, the illumination distance and the illumination time. Er³⁺:BGZ phosphors are promising UVemitting phosphors for healthy indoor illumination.     

Capillary isoelectric focusing of proteins with liquid core waveguide laser-induced fluorescence whole column imaging detection

A capillary isoelectric focusing (CIEF) system with liquid core waveguide (LCW) laser-induced fluorescence whole column imaging detection was developed in this study. A Teflon AF 2400 capillary was used as both the separation channel and the axially illuminated LCW. The excitation light was introduced at one end of the capillary, and propagated forward within the capillary. As the Teflon AF 2400 capillary has a refractive index (n = 1.29-1.31) lower than that of water (n = 1.33), total internal reflection was very apparent The employment of the Teflon AF 2400 capillary avoided the use of high refractive index additives such as glycerol, accommodating the system to wider applications. Due to its inert chemical properties, the capillary exhibited limited protein adsorption and electroosmotic flow; thus, the need for capillary preconditioning with polymeric solution and the addition of polymeric additives into the sample mixture can be avoided. Three types of proteins, naturally fluorescent proteins, covalently labeled proteins, and noncovalently labeled proteins, were examined using this method. CIEF under denaturing conditions was also explored, and several advantages over the native mode were found. When compared to a commercially available instrument with UV detection, the separation efficiency and peak capacity were similar while the detection sensitivity was enhanced by 3-5 orders of magnitude.     

NIR‐II‐Excited Intravital Two‐Photon Microscopy Distinguishes Deep Cerebral and Tumor Vasculatures with an Ultrabright NIR‐I AIE Luminogen

Intravital fluorescence imaging of vasculature morphology and dynamics in the brain and in tumors with large penetration depth and high signal‐to‐background ratio (SBR) is highly desirable for the study and theranostics of vascular‐related diseases and cancers. Herein, a highly bright fluorophore (BTPETQ) with long‐wavelength absorption and aggregation‐induced near‐infrared (NIR) emission (maximum at ≈700 nm) is designed for intravital two‐photon fluorescence (2PF) imaging of a mouse brain and tumor vasculatures under NIR‐II light (1200 nm) excitation. BTPETQ dots fabricated via nanoprecipitation show uniform size of around 42 nm and a high quantum yield of 19 ± 1% in aqueous media. The 2PF imaging of the mouse brain vasculatures labeled by BTPETQ dots reveals a 3D blood vessel network with an ultradeep depth of 924 µm. In addition, BTPETQ dots show enhanced 2PF in tumor vasculatures due to their unique leaky structures, which facilitates the differentiation of normal blood vessels from tumor vessels with high SBR in deep tumor tissues. Moreover, the extravasation and accumulation of BTPETQ dots in deep tumor (more than 900 µm) is visualized under NIR‐II excitation. This study highlights the importance of developing NIR‐II light excitable efficient NIR fluorophores for in vivo deep tissue and high contrast tumor imaging.     

Grating-coupling of surface plasmons onto metallic tips: a nanoconfined light source

We describe and demonstrate a new nanometer-scale broadband light source. It is based on the grating-coupled excitation of surface plasmon polaritons (SPPs) on the shaft of a sharp conical metal taper with a tip radius of few tens of nanometers. Far-field excitation of linear nanoslit gratings results in the resonant generation of SPPs traveling over more than 10 mum to the tip apex and converging to an intense radiative local light spot. Such nanofabricated tips are expected to find various applications in nanospectroscopy, overcoming problems with background illumination in apertureless microscopy.