Search

In this article authors present a prototype of a dichroic scanning nonlinear optical microscope with a photodetecting polymer Scanner and a customized objective lens with an external back focal plane. Two types of two-axis electromagnetic actuated polymer scanners are manufactured by a standard printed circuit board (PCB) fabrication process. Both scanners share the same overall dimensions of 45 mm by 45 mm. The maximum optical scan angle of a type I scanner is 13 degrees at 420 Hz by the fast axis in resonance and 4 degrees by the slow axis in the linear mode. The type II scanner shows similar performance. A scan lens as well as a tube lens in a conventional nonlinear optical microscope are replaced by placing such a polymer scanner on the external back focal plane of a customized water immersion objective lens. The objective lens has the back working distance of 6 mm, the numerical aperture of 0.7, the front working distance of 1.16 mm, a field of view of 1.67 mm in the diameter and the angle of view of +/−15 degrees. Preliminary imaging results are also presented.

Properties regarding stratum corneum (SC), the outermost membrane of the skin, remain an active area in dermatologic and cosmetic research. The reduced thickness of SC is associated with varied adverse statuses such as skin lipid deficiency, skin barrier dysfunctions and skin deceases, etc. Emulsifiers with existing irritative effects on skin components also face the risk of decreasing SC thickness. We have been focusing on the effects of PEGylated emulsifiers on the skin and have an interest in finding the role of their polyethylene glycol (PEG)-chain length in tuning skin irritations. With this aim, PEG-stearyl ethers with different numbers of hydrophilic chains were applied on the skin, and their influence on skin thickness was discovered to determine their skin barrier effect. Confocal Raman spectroscopy (CRS) with extensive application in skin research was used here. To obtain the precise determination of skin thickness, our secondary aim was to find the optimal CRS configuration referring to varied objectives and pinhole sizes where further study is still in demand. Therefore, SC thickness measured via eddy current approach served as reference. The applied PEG-stearyl ethers formed the system to achieve varied thicknesses. Results confirmed that the skin interactions rose with increasing PEG-chain length, however only up to a certain limit, with decreasing effects recorded from PEG-40 stearyl ether and no effects observed from PEG-100 stearyl ether. Simultaneously, CRS combined with water immersion objective and 50 μm pinhole presented the most consistent values to the references and exhibited better spectral intensity and signal-to-noise ratio. Correlation plots involving different cases of configurations were calculated for error corrections. Taken together, this work helps to identify the potential mechanisms governing the interactions between PEG-stearyl ethers and skin and offers powerful evidence of using CRS as a reliable alternative to obtain accurate thickness values.

Plastic films of multiple layers are widely used in packaging. While a detailed spatially resolving analysis is highly relevant, the currently used approaches are either tedious or lack spatial resolution. An advanced method of confocal Raman microscopy was therefore developed in this work, using a water immersion objective combined with multivariate data analysis and ray tracing models for analysing a commercial-grade five-layer plastic film. The water immersion objective and ray tracing models are able to reduce the refraction induced artefacts in depth profiling and ensure a correct and accurate interpretation of each layer thickness. The multivariate data analysis can distinguish each layer without prior knowledge of the sample and unique markers of each composition. Finally, the resulting thicknesses of the film layers could be successfully compared to complementary experimental results. In conclusion, each layer thickness of a multilayer polymer film and its chemical composition could be measured accurately and non-invasively without sample preparation.

Recent development of visible light Optical Coherence Microscopy (i.e. Optical Coherence Tomography employing high numerical aperture Objective) has pushed the axial/transverse resolution limit to < 2 μm within tissues. Here, a fiber-based visible light Fourier domain (FD) Optical Coherence Microscopy system is presented, employing a 10x water immersion objective to achieve higher transverse resolution and reduced specular reflection when imaging human skin in vivo, using ~ 1-2 mW of incident power. Though with limited penetration depth, we demonstrated its application for in vivo imaging of human skin, and further measured the thicknesses of epidermis and dermo-epidermal junction. Furthermore, the total attenuation coefficient of the skin tissues was also estimated across epidermis and dermis.

Bridging veins (BVs) drain the blood from the cerebral cortex into dural sinuses. BVs have one end attached to the brain and the other to the superior sagittal sinus (SSS), which is attached to the skull. Relative movement between these two structures can cause BV to rupture producing acute subdural haematoma, a head injury with a mortality rate between 30 and 90%. A clear understanding of the BVs microstructure is required to increase the biofidelity of BV models when simulating head impacts. Twelve fresh BV samples draining in the superior sagittal sinus (SSS) from a single human cadaver were cut open along their length and placed on an inverted multiphoton microscope. To ensure that the BVs were aligned with the axial direction an in-house built, uniaxial tension set-up was used. Two scans were performed per sample. Before the first scan, a minor displacement was applied to align the tissue; then, a second scan was taken applying 50% strain. Each BV was scanned for a length of 5 mm starting from the drainage site into the SSS. Imaging was performed on a Zeiss LSM780 microscope with an 25[Formula: see text] water immersion objective (NA 0.8), coupled to a tunable MaiTai DS (Spectraphysics) pulsed laser with the wavelength set at 850 nm. Second harmonic and fluorescence signals were captured in forward and backward direction on binary GaAsP (BiG) detectors and stored as four colour Z-stacks. Prior to the calculation of the local orientations, acquired Z-stacks were denoised and enhanced to highlight fibrillar structures from the background. Then, for each Z-plane of the stack, the ImageJ plugin OrientationJ was used to extract the local 2D orientations of the fibres based on structure tensors. Two kinds of collagen architectures were seen. The most common (8[Formula: see text]12 samples) was single layered and had a uniform distribution of collagen. The less common (4[Formula: see text]12 samples) had 2 layers and 7 to 34 times thicker collagen bundles on the outer layer. Fibre angle analysis showed that collagen was oriented mainly along the axial direction of the vessel. The von Mises fittings showed that in order to describe the fibre distribution 3 components were needed with mean angles [Formula: see text] at [Formula: see text] 0.35, 0.21, [Formula: see text] 0.02 rad or [Formula: see text] 20.2[Formula: see text], 12.1[Formula: see text], [Formula: see text] 1.2[Formula: see text] relative to the vessel's axial direction which was also the horizontal scan direction. ispartof: BIOMECHANICS AND MODELING IN MECHANOBIOLOGY vol:19 issue:6 pages:2455-2489 ispartof: location:Germany status: published

Optical whispering-gallery-mode (WGM) microresonator-based sensors with high sensitivity and low detection limit down to single unlabeled biomolecules show high potential for disease diagnosis and clinical application. However, most WGM microresonator-based sensors, which are packed in a microfluidic cell, are a &ldquo, closed&rdquo, sensing configuration that prevents changing and sensing the surrounding liquid refractive index (RI) of the microresonator immediately. Here, we present an &ldquo, open&rdquo, sensing configuration in which the WGM microdisk laser is directly covered by a water droplet and pumped by a water-immersion-objective (WIO). This allows monitoring the chemical reaction progress in the water droplet by tracking the laser wavelength. A proof-of-concept demonstration of chemical sensor is performed by observing the process of salt dissolution in water and diffusion of two droplets with different RI. This WIO pumped sensing configuration provides a path towards an on-chip chemical sensor for studying chemical reaction kinetics in real time.

Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope’s resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5–180 μm.

Most flying-spot Optical Coherence Tomography (OCT) and Optical Coherence Microscopy (OCM) systems use a symmetric confocal geometry, where the detection path retraces the illumination path starting from and ending with the spatial mode of a single mode optical fiber. Here, we describe a visible light OCM instrument that breaks this symmetry to improve transverse resolution without sacrificing collection efficiency in scattering tissue. This was achieved by overfilling a 0.3 numerical aperture (NA) water immersion objective on the illumination path, while maintaining a conventional Gaussian mode detection path (1/e2 intensity diameter ~0.82 Airy disks), enabling ~1.1 μm full-width at half-maximum (FWHM) transverse resolution. At the same time, a ~0.9 μm FWHM axial resolution in tissue, achieved by a broadband visible light source, enabled femtoliter volume resolution. We characterized this instrument according to paraxial coherent microscopy theory, and then used it to image the meningeal layers, intravascular red blood cell-free layer, and myelinated axons in the mouse neocortex in vivo through the thinned skull. Finally, by introducing a 0.8 NA water immersion objective, we improved the lateral resolution to 0.44 μm FWHM, which provided a volumetric resolution of ~0.2 fL, revealing cell bodies in cortical layer I of the mouse brain with OCM for the first time.

Significance Cryofluorescence imaging is of great interest in biological microscopy because vitrified samples (i.e., frozen without ice crystallization) are free from fixation artifacts, are highly photostable, and allow direct correlation with electron cryomicroscopy. Here, we show a concept for conducting cryo-light microscopy in immersion that provides a twofold to fivefold increase in image brightness and higher resolution than are attainable with air objectives. Spherical aberration at an imaging temperature below the glass transition of water (−135 °C) is corrected by our approach., Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at −140°C.

Most flying-spot optical coherence tomography and optical coherence microscopy (OCM) systems use a symmetric confocal geometry, where the detection path retraces the illumination path starting from and ending with the spatial mode of a single-mode optical fiber. Here we describe a visible light OCM instrument that breaks this symmetry to improve transverse resolution without sacrificing collection efficiency in scattering tissue. This was achieved by overfilling a water immersion objective on the illumination path while maintaining a conventional Gaussian mode detection path (1/e2 intensity diameter ∼0.82 Airy disks), enabling ∼1.1 μm full width at half-maximum (FWHM) transverse resolution. At the same time, a ∼0.9 μm FWHM axial resolution in tissue, achieved by a broadband visible light source, enabled femtoliter volume resolution. We characterized this instrument according to paraxial coherent microscopy theory and, finally, used it to image the meningeal layers, intravascular red blood cell-free layer, and myelinated axons in the mouse neocortex in vivo through the thinned skull.

The role of the immersion medium in the microscale speherical lens imaging is studied. We find that when a microsphere lens is semi-immersed in the SU-8 resist, both the microsphere and the SU-8 resist can work as a lens. The microsphere lens forms the images from the small-k Fourier components of an object, and this image formation does not depend on the immersion of the SU-8 resist. The SU-8 resist can work as a lens and form the images from the large-k Fourier components. When the distance d between the focal spot of the SU-8 lens and the object is close, the clear large-k Fourier components image can be observed even without the microsphere.

A woofer–tweeter adaptive optical structured illumination microscope (AOSIM) is presented. By combining a low-spatial-frequency large-stroke deformable mirror (woofer) with a high-spatial-frequency low-stroke deformable mirror (tweeter), we are able to remove both large-amplitude and high-order aberrations. In addition, using the structured illumination method, as compared to widefield microscopy, the AOSIM can accomplish high-resolution imaging and possesses better sectioning capability. The AOSIM was tested by correcting a large aberration from a trial lens in the conjugate plane of the microscope objective aperture. The experimental results show that the AOSIM has a point spread function with an FWHM that is 140 nm wide (using a water immersion objective lens with NA=1.1) after correcting a large aberration (5.9 μm peak-to-valley wavefront error with 2.05 μm RMS aberration). After structured light illumination is applied, the results show that we are able to resolve two beads that are separated by 145 nm, 1.62× below the diffraction limit of 235 nm. Furthermore, we demonstrate the application of the AOSIM in the field of bioimaging. The sample under investigation was a green-fluorescent-protein-labeled Drosophila embryo. The aberrations from the refractive index mismatch between the microscope objective, the immersion fluid, the cover slip, and the sample itself are well corrected. Using AOSIM we were able to increase the SNR for our Drosophila embryo sample by 5×.

The 1700-nm window has emerged as a promising excitation window for multiphoton microscopy (MPM). On one hand, the combined low tissue absorption and scattering make this window well suited for deep-tissue MPM; on the other hand, the long excitation wavelength makes higher-order MPM in biological tissues feasible, e.g., recently 4-photon fluorescence MPM in mouse brain has been demonstrated. Objective lens is a key optical component in the entire MPM setup. Multiphoton signal levels are largely dependent on the transmittance of objective lens. Here we demonstrate experimental results of transmittance measurement of two water immersion objective lenses commonly used for MPM at the 1700-nm window, covering both the excitation and the signal window. Our target application is MPM of even higher order excited at this window, i.e., 4th harmonic generation (FHG) imaging and 5-photon fluorescence generation. Our results show that, although the customized objective lens offers higher transmittance at the excitation window, it suffers from dramatically degraded transmittance at the signal window, compared with the non-customized objective lens. These results will offer guidelines for selection of proper objective lens for higher-order MPM at the 1700-nm window.

The angular spectrum of radially polarized Laguerre–Gaussian beams was tailored by an annular aperture in a semiconductor solid immersion lens microscope. The radii of two concentric rings in the amplitude aperture were optimized by a multi-objective particle swarm optimization algorithm. A GaAs solid immersion lens with NA=3.4 was used in the numerical calculation and a spot size of 98 nm ( λ / 10 ) at a wavelength of 1064 nm illumination was obtained.

The optical parameters of temporal-focusing multiphoton excitation microscopy (TFMPEM), which is capable of achieving varying wavelength excitation for multiple fluorophore measurement, was systematically examined to have good excitation performance. For this purpose, the approaches were adopted to quantitatively evaluate the grating groove density, focal length of the collimating lens and objective, and different excitation wavelengths. A grating with a groove density of 830 lines/mm enables the TFMPEM system to achieve a wavelength range of 700–1000 nm by adjusting the incident angle of the ultrafast laser on the grating; a diffraction efficiency of 81 ± 3% was obtained at this wavelength range. By using the 830 lines/mm grating, a collimating lens with a 500 mm focal length and a 60× water immersion objective, we achieved a large excitation area and a better filling effect of the spectrum band of the pulse on the back focal plane of the objective; these parameters ensure high optical sectioning and small variation in the illumination power density within this wavelength range. The variation ranges of the excitation area, optical sectioning, and illumination power density of 4272 ± 798 μm2, 2.6 ± 0.3 μm, and 71 ± 29%, respectively, were obtained in two-photon excitation fluorescence imaging at 700–1000 nm excitation wavelengths.

A new confocal measurement for lens refractive index and thickness is proposed based on the property that the peak of an axial intensity curve precisely corresponds to the focus of the objective in a confocal system, which uses the peak of confocal axial response curve to precisely identify the positions of the objective when the measurement pencil is focused on the vertex of the test lens and the reflector with or without the test lens in the measurement light-path, and then uses ray tracing facet iterative calculation to obtain the refractive index and thickness of the test lens simultaneously. The preliminary experiments indicate that the measurement accuracy can reach about 3.8 × 10 −4 for the refractive index and 0.05% for the thickness.

Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and “video-mosaicing”, extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

A tunable-focus liquid lens using an iris diaphragm is demonstrated. In the central opening area, two immiscible liquids with different refractive indices meet, resulting in a curved shape with a contact angle on the blade surface of the diaphragm. The liquids with a curved shape exhibit a lens character. By changing the aperture of the diaphragm, the shape of the liquid is changed accordingly which, in turn, leads to tunable focal length. Based on this operation approach, the focal length (f) of our lens could be tuned in the range of 13 ≤ f ≤ 105 cm when its aperture is changed from 1.3 to 3.2 mm. At f = 105 cm, the resolution of our lens can resolve over 45 line pairs per millimeter at room temperature. Our liquid lens, by changing aperture size, has the advantages of compact structure, easy operation, large optical power, and multistability.

We investigate the defocus and image quality affected by a dielectric interface on high numerical aperture focusing of linearly polarized illumination in aplanatic mode. Theoretical and experimental demonstration is performed on subsurface backside microscopy of silicon integrated circuits, showing that the high longitudinal magnification provided by solid immersion lens microscopy allows the observation of significant astigmatism. It is shown that a 50 micron longitudinal displacement of the objective lens with respect to the sample is necessary to achieve maximum resolutions in two directions.

A high power cw laser beam propagating in an absorbing medium spreads, distorted or deflected as a result of thermally self-induced refractive index gradients. In this paper, optical characteristic of thermal lens effect in liquid ethanol was studied experimentally. The thermal lens was induced by focusing of a high power He–Ne laser beam in ethanol (absorbing liquid). The thermal lens effect was investigated by Z-scan method. Depending on the focusing position of the beam in liquid cell, the results show both intensity increasing and decreasing on a screen, which is fixed at a specific position with respect to the cell. The results show such effects depend on the lens focal length, the liquid cell size, and the focusing position of the lens. According to the results there is a competition between focusing and defocusing of the transmitted beam if the position of the focusing region inside the sample cell is changed. Using a simple optical description, the experimental results were interpreted.

Liquid renovation is perhaps the best method to reduce contamination in immersion lithography, but it brings lens distortion because of the high density and viscosity of the immersion liquids compared to air. In this paper, considering wafer scanning, the three-dimensional computational liquid dynamics model and finite-element model are developed to investigate the lens distortion for immersion liquid renovation. After analyzing the influence of wafer scanning directions, the empirical formulas of the lens distortion for three-dimensional model are established to describe the effect of the parameters of lens and polymer which are used to mount lens and the parameters of immersion liquid flow on lens distortion.

A compact, high-speed variable-focus liquid lens using acoustic radiation force is proposed. The lens consists of an annular piezoelectric ultrasound transducer and an aluminum cell (height: 3 mm; diameter: 6 mm) filled with degassed water and silicone oil. The profile of the oil-water interface can be rapidly varied by applying acoustic radiation force from the transducer, allowing the liquid lens to be operated as a variable-focus lens. A theoretical model based on a spring-mass-dashpot model is proposed for the vibration of the lens. The sound pressure distribution in the lens was calculated by finite element analysis and it suggests that an acoustic standing wave is generated in the lens. The fastest response time of 6.7 ms was obtained with silicone oil with a kinematic viscosity of 100 cSt.

A structured lens with a large numerical aperture for high diffraction efficiency is proposed. This structured lens is formed by varying high-refractive-index-dielectric (HRID) square holes in a metallic film. Compared with previous structured lenses formed by varying air square holes, the modulated wavefront at the exit pupil of the structured lens is smoother and the diffraction efficiency is greatly improved. A dielectric film was introduced as a buffer layer to reduce the energy loss at the interface caused by the great impedance mismatching between air and the high refractive index dielectric. A lens with an aperture of 100 µm and a focal length of 50 µm was designed; its diffraction efficiency has been raised from 22.8% to 47.2% by the finite difference and time domain (FDTD) algorithm. This structured lens has the advantages of not only light weight and high compactness, but also large numerical aperture, high diffraction efficiency, and good imaging quality.

The aim of this study was to analyze and evaluate the oral mucosa and the surface constitution of dental materials to display the characteristics and the behaviour of dental composites concerning the margin fit. Therefore, the gingiva and composite restorations of 45 patients aged 17-69 years were examined with a new digital confocal laser scanning microscope developed in Rostock. The device works in contact with the surface of the examination region using a water immersion objective and a diode laser with a wavelength of 670 nm. As a result of a PMMA-Cap with a thickness of 0.5 mm, we can create images with exact depth information and of very good quality. The epithelium of the gingiva with its cellular and subcellular structures could be observed up to a depth of 200 microm. Also, papillary crests, blood vessels and even the blood flow were visible. An inflammation of the gingiva could be verified because of the existence of inflammatory cells. The enamel and dental restorations could be observed up to a depth of 80 microm and allowed the evaluation of the margin fit. Confocal laser scanning microscopy seems to be suitable for the non-invasive examination of oral mucosa and dental materials in vivo.

This paper describes a simple method for fabricating a variable-focus lens using polydimethylsiloxane (PDMS) filling with liquid to produce a variable-focus lens. A 2-mm diameter lens was designed in this experiment, expected to reach a focal length in the range of 3 ∼ 12 mm. The theoretical value between the liquid volume and the lens contact angle at different focal lengths were simulated and measured. The pumped-in liquid volumes ranged from 200 to 1,400 μl. The contact angles ranged from 14.25° to 49.02°. Variable focal length was produced by changing the PDMS film deformation using different micro-fluidic volumes. The focal length produce in the experiment was from 4 ∼ 10 mm. The proposed method successfully fabricated a variable-focus lens. Bonding PDMS only once using no expensive instrument such as oxygen plasma was accomplished. The final objective is to insert the variable focus lens into portable optical imagery products.

We advocate the use of a polymeric cover layer for protecting the data layer and the tip of the solid immersion lens in near-field optical recording system. With a cover on top of the data layer, the numerical aperture (NA) of the objective lens is limited to the refractive index of the cover material. This means that the maximum attainable NA of cover-incident near-field systems and therefore the maximum achievable storage density is lower compared to that for first surface systems. This lower storage capacity per layer can be more than compensated for by using multiple data layers which is not possible in first-surface systems with bare discs. In this paper we present first experimental results for near-field recording with a solid immersion lens that focuses through a cover layer and a spacer layer onto a data layer, as in a dual-layer near-field disc.

A technique for measuring the refractive index of a lens is described in this paper. In the present paper, an innovative technique has been proposed in which acousto-optics is involved. A new formula for evaluating the refractive index of a lens glass material is reported which is independent of lens parameters. A rectangular glass cell filled with liquid has been used as a photo-sonar device, which also has a piezoelectric crystal for generating ultrasonic waves. The test lens is immersed inside the ultrasonic cell. The focal length of the test lens has been varied by immersing it inside the ultrasonic cell filled with known liquid. The laser light travelling through the lens inside the glass cell gets diffracted when the ultrasonic waves are generated. Thus an interaction between sound and light wave occurs, resulting in a grating-like action, and the diffraction dots are displayed on a screen. A relation between the refractive index of liquids and the velocity of ultrasonic waves has been derived which also involves the separation of diffraction dots.

The liquid lens has a broad application prospect in zoom and image systems for its quick corresponding speed, small volume and low consumption. However, the range of focal length is small, because the refractive index of conductive liquid has a little difference to that of non-conductive liquid in the liquid lens. In order to increase the zoom range of the liquid lens, a composite lens made up of a liquid lens and a solid lens is presented in this paper. The focal length variation property of the composite lens has been investigated, and the corresponding composite lens parameters are optimized. The zoom of composite lens with different slope solid interfaces has been studied.

We developed solid immersion lenses made of a KTaO3 monocrystal. The refractive index of KTaO3 is 2.382 at a wavelength of 405 nm. Using KTaO3 as the raw material of a solid immersion lens, we could design an effective numerical aperture of 2.20. We observed an eye pattern of a 150 GB capacity with a 130 nm track pitch and a 47.6 nm bit length. The areal density is 104.3 Gbit/in.2.

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 microm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water-polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.

Chipmakers are turning to water as the way to delay a massively expensive and difficult transition to lithography based on soft X-rays. The overall resolution of the optical system is defined by the wavelength of the illuminating light divided by the numerical aperture (NA) of the lens system; the higher the NA value, the greater the possible resolution. The NA of the illumination optics is defined by the sharpness of the cone of light that is focused by the final lens onto the wafer and the refractive index of the material it passes through on the way. One way to increase the NA and thus the overall resolution is to use a medium between the lens and the wafer with a refractive index that is higher than air's value of 1. Water, with a refractive index of 1.33, pushes up the NA of the lens system and also helps avoid internal reflections in the lens and reflections from the resist. Going from dry lithography to wet lithography will improve minimum resolutions. It also reduces the angle of incidence and therefore increases the depth of focus.

An innovative approach for determining the refractive index of lenses has been reported. The lens as a specimen remains safe because neither it is touched nor any part of it is removed for experimentation. All the serious difficulties associated with refractive index measurements of glass material in the form of a lens have been set aside. For this purpose, the lens immersed in a liquid inside a glass cell has been used. Laser light is incident on Fabry–Perot (F–P) etalon, which forms the fringes in transmission side. This fringe pattern of circular rings is brought to the back focal plane of lens immersed in a liquid. A new mathematical formula, involving the diameter of interferometric fringes and refractive indices of liquids, is required for evaluating the refractive index of a glass sample in the form of a lens, have been deduced.

We have investigated high-density near-field readout using a solid immersion lens with a high refractive index. By using a glass material with a high refractive index of 2.08, we developed an optical pick-up with the effective numerical aperture of 1.8. We could observe a clear eye pattern for a 50 GB capacity disc in 120 mm diameter. We confirmed that the near-field readout system is promising method of realizing a high-density optical disc system.

For many laser applications, information on irradiated laser power is important. However, direct measurement of laser power through a high numerical aperture objective lens is difficult in a laser microscope. In this article, we propose a method which use a solid immersion lens (SIL) for such measurements. A laser beam focused by an objective lens is introduced to the flat surface of a SIL, emitted through the spherical surface, and then detected. In this way the divergence of the laser beam is reduced, and as a result the detection efficiency of the laser power increases. From theoretical analysis, a Weierstrass-sphere type SIL was found to be an appropriate thickness for this propose. Transmittance of the SIL is evaluated for several refractive indeces. The validity of this method is confirmed experimentally.

A new infrared confocal refractive index measurement (IR-CRIM) method with high precision is proposed for a lens. Based on the property that the maximum point of a confocal axial intensity response curve accurately corresponds to the converging focus of the measuring beam, IR-CRIM can precisely identify the front and back vertices of the test lens, and obtain the optical thickness d of the test lens, and resulting calculate the lens refractive index n using the ray-tracing algorithm. The broadband refractive index dispersion of the test lens can be acquired by the Cauchy dispersion law and the n obtained at several wavelengths. Preliminary experimental results and theoretical analyses indicate that IR-CRIM achieves an accuracy of 6 × 10−4 in the wavelength range of 500–1700 nm. It provides a novel approach for the high-precision and direct measurement of the refractive index of a lens in visible and near infrared wavelength range.

Summary This paper sets out to establish resolution limits for electron objective immersion lenses that can be used for high resolution scanning electron microscopy. On-axis aberration coefficients, both spherical (Cs) and chromatic (Cc), are calculated for immersion lenses that are: purely magnetic, of the electric retarding field type, and that have mixed electric-magnetic fields. The aperture lens model is used to derive a general framework by which these lenses can be compared. Simulation results show that the mixed field electric-magnetic immersion lens is expected to provide the best image resolution. For a primary beam landing energy of 1 keV, and for field strength limits set by magnetic saturation and vacuum electrical field breakdown, the lowest on-axis aberration coefficients predicted for this lens lie between 50 μm to 60 μm.

Metal-dielectric core-shell particles represent promising tools in nanoplasmonics. In combination with optical tweezers they can be manipulated in a contactless way through fluid and their plasmonic properties can be used to probe or modify the local environment. We perform a numerical parametric study to find the particle geometry and material parameters under which such particle can be stably confined in optical tweezers. We use the theory based on Mie scattering in the focal field of an ideal water immersion objective of numerical aperture NA=1.2. For very thin metal layers we find that strong trapping on the optical axis can be achieved.

High resolution laser imaging, using high numerical aperture (NA) solid immersion lens (SIL) for backside fault isolation imposes stringent sample preparation requirements; as a result of the short focal length of SIL, a die must be thinned to a targeted thickness with less than a ±5 μm silicon thickness variation across the entire die. Flip chip packaged dice suffer from warpage due to various package sizes and substrate thicknesses. Such broad spectrums of part geometries pose a great challenge to meet such silicon planarity requirements. As relaxation of the packaged silicon during polishing causes the warpage profile to change dynamically and unpredictably throughout the thinning process, it has become an added challenge to meet the stringent sample preparation requirements. To overcome the stochastic nature of this problem, a two-step polishing recipe consisting of computer numerical control (CNC) mechanical milling and polishing processes has been developed to achieve sufficient silicon thickness uniformity to enable SIL imaging across an entire silicon chip as large as approximately 20 mm x 15 mm.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.

Nano-scale resolution in miniature optical systems has been realized in the optical data storage industry. Numerical apertures greater than unity have been achieved in by utilizing the high index material of a hemispherical Solid Immersion Lens (SIL), which increases the resolution of the backing objective by a factor that is related to the refractive index of the SIL. In this research, a custom Hyper-Blu-Disc (HBD) NA=1.4 SIL objective is utilized for high-fidelity readout of data pits beneath a 100μm thick cover layer on an optical Blu-Ray Disc. If realized commercially, the increase in data density could be 3X today’s Blu-Ray technology. A distinct difference between this work and other work with SILs in optical data storage is the relatively thick cover layer of 100μm. Recently, there has been interest in discovering new ways to apply the technology and methods used in optical data storage for other means. The inherent design of the HBD objective to image through a shallow layer of dielectric material may lend itself to be used as an effective means for characterizing subsurface damage in optical materials. This research will furthermore investigate the HBD objective as a means of detecting subsurface damage.

We present the microfabrication of a solid immersion lens from silicon for scanning near-field optical microscopy. The solid immersion lens (SIL) achieves spatial resolution better than the diffraction limit in air without the losses associated with tapered optical fibers. A 15-/spl mu/m-diameter SIL is formed by reflowing photoresist in acetone vapor and transferring the shape into single-crystal Si with reactive ion etching. The lens is integrated onto a cantilever for scanning, and a tip is fabricated opposite the lens to localize lens-sample contact. Using the Si SIL, we show that microfabricrated lenses have greater optical transparency and less aberration than conventional lenses by focusing a plane wave of 633-nm light to a spot close to a wavelength in diameter. Microlenses made from absorbing materials can be used when the lens thickness Is comparable to the penetration depth of the light. Tolerance to errors in curvature and thickness is improved in micromachined lenses, because spherical aberrations decrease with lens diameter. We demonstrate scanning near-field optical microscopy with the Si SIL and achieve spatial resolution below the diffraction limit in air by resolving 200-nm lines with 633-nm light.

Microscopic inspection of heterogenous three-dimensional (3D) objects such as oral implants, or implants in general, is conventionally performed either on ground sections of methyl-metacrylate-embedded material, at the cellular level by histologic analysis of the peri-implant tissue by light microscopy (LM), or at the supramolecular level by transmission electron microscopy (TEM). Alternatively, the architecture of the tissue/implant interface is visualized by scanning electron microscopy (SEM). The two approaches exclude each other because of the sample preparation.We elaborate conditions for the non-invasive analysis of tissue/implant interfaces by confocal laser scanning microscopy (CLSM) in buffer, hoping to obtain a 3D view of fluorescently labeled tissue constituents at the tissue implant interface and, through subsequent SEM, of the metal surface. The use of water-immersion objectives, originally developed for high LM under physiological conditions is essential. In an exploratory approach, the tissue/Ti-interfaces of two retrieved dental implants were analyzed. One was a step-cylinder used for orthodontic anchoring and the other was an endosseous step-screw implant retrieved after infection-related loosening prior to load. The adhering tissue fragments were fluorescently triple-labeled for actin, fibronectin, and sm-alpha-actin. Optical sections for fluorescent images and for the laser reflection map were registered concomitantly. This approach allowed the labeled structures to be located on the metal surface. Subsequently, the same implants were prepared for SEM of the tissue/implant interface, and upon removal of the adhering structures, of the underlying metal surface. Thus, specific proteins can be identified and their spatial architecture as well as that of the underlying metal surface can be visualized for one and the same implant. The immediate visualization after fluorescence labeling in buffer by means of water immersion objective lenses proved most critical.

The method of using immersion medium to correct spherical aberration for water immersion objectives when the samples are not water is investigated. Spherical aberration is measured by an interferometer converted from a confocal microscope for samples with different refractive indices. When the proper refractive index of the immersion medium and thickness of cover slip are selected, the measured spherical aberration approaches zero. A theoretical model can be used for prediction of the immersion medium to correct spherical aberration for various samples. Using the thinnest available cover slip (100 microm), the zero spherical aberration condition can be applied to samples with refractive index as high as 1.40. Confocal images in the condition of almost no spherical aberration are included to demonstrate the improvement of axial resolution due to this correction.

The in-depth imaging properties of two-photon excitation microscopy were investigated and compared with those of confocal microscopy. Confocal imaging enabled the recording of images from dental biofilm down to a depth of 40 microm, while two-photon excitation images could be recorded at depths greater than 100 microm. Two-photon excitation point spread functions (PSFs) were recorded at depths ranging from 0 to 90 microm depth using 220-nm diameter fluorescent beads immersed in water. PSFs were measured using both a high numerical aperture oil immersion objective and a water immersion objective. The experiments carried out using the oil immersion objective showed a rapid degradation of both the axial and lateral resolution due to spherical aberrations. In addition, the detected fluorescence intensity rapidly decreased as a function of depth. The experiments carried out using the water immersion objective showed no significant degradation of both the axial and lateral resolution and the fluorescence intensity.

High-density magneto-optical recording using near field optics with a solid immersion lens is discussed. The spot size, and defocusing distance are calculated for various combinations of n and NA. Also, the misalignment of the objective lens and SIL is discussed. The magnetic and magneto-optical properties of SIL media candidates of TbFeCo/Pt multilayers are presented.

We investigated high-density near-field readout using a diamond solid immersion lens (SIL). A synthetic single-crystal chemical vapor deposition diamond provides a high refractive index and a high transmission for a wide wavelength range. Since the refractive index at a wavelength of 405 nm is 2.458, we could design a solid immersion lens with an effective numerical aperture of 2.34. Using the diamond SIL, we observed the eye pattern of a 150-GB-capacity (104.3 Gbit/in.2) disk with a track pitch of 130 nm and a bit length of 47.6 nm.

Using the immersion lensing technique, the resolution of a conventional spherical lens can be improved by a factor of 1/n over its value in air (n, the refractive index of the immersion medium). Depending on the relative position between an object and a lens, either a real or a virtual image is formed. Here we report a new physical phenomenon experimentally observed in the microscale lens imaging. We find that when a microscale spherical lens is semi-immersed in a medium, the resolution of the lens is improved as it can intercept more fine details of the object. However, the microscale lens has two image channels for the fine and coarse details and two images corresponding to the two components can be formed simultaneously. Our findings will advance the understanding of the super-resolution imaging mechanisms in microscale lenses.

Due to some physical limitations of conventional condensers, inverted compound microscopes are not optimally suited for imaging living brain slices with transmitted light. Herein is described a simple device that converts an inverted microscope into an effective tool for this application by utilizing an objective as a condenser. The device is mounted on a microscope in place of the condenser, is threaded to accept a water immersion objective, and has a slot for a differential interference contrast (DIC) slider. When combined with infrared video techniques, this device allows an inverted microscope to effectively image living cells within thick brain slices in an open perfusion chamber.

A modified optical system for the light microscope has been devised in order to remotely shift the focal plane and to manipulate the point spread function for any given objective lens. An adjustable telescope system is inserted into the microscope tube so as to move the intermediate image position, thus achieving two goals of fundamental importance for the three-dimensional imaging of biological samples. First, it allows the focus to be rapidly varied without actually moving the objective lens. This permits high throughput three-dimensional microscopy of living specimens. Secondly, it makes possible the compensation of objective lens spherical aberration. This distortion is especially significant when high numerical aperture objectives are utilized to image deep into thick specimens.