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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.

Electrochemical tip-enhanced Raman spectroscopy (EC-TERS) appears as a promising in situ nanospectroscopic tool for characterization and understanding of the electrochemical interfacial processes at the nanometer scale and molecular level. However, the wide application of EC-TERS is hampered by its low sensitivity as a result of the optical path distortion due to the refractive index mismatch of the multilayer media (air, glass, and electrolyte). Here, we propose a new side-illumination EC-TERS setup by coupling a water immersion objective with a high numerical aperture to a scanning tunneling microscope scanning head customized with a large open space and a compact spectroelectrochemical cell. It not only effectively eliminates the optical distortion but also increases the sensitivity remarkably, which allows sensitive monitoring of the electrochemical redox processes of anthraquinone molecules. More importantly, EC-TERS is able to independently control the tip position and laser illumination position. By utilizing this feature, we reveal that the irreversible reduction reaction of anthraquinone observed in EC-TERS is induced by the synergistic effect of the negative potential and laser illumination rather than the localized surface plasmon. The highly improved sensitivity and the flexibility to control the tip and laser illumination position on the nanometer scale endows EC-TERS as an important tool for the fundamental understanding of the photo- or plasmon electrochemistry and the interfacial structure-activity relationship of important electrochemical systems.

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.

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 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.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has a diameter of several microns, and the release sites positioned outside of the electrode rim do not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

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 present protocol to visualize living bacteria at the pore level of cauliflower hydathodes is simple and trained users in confocal microscopy can execute it successfully. It can be easily adapted to capture images with other plant-microorganism interactions at the leaf surface and should be useful to obtain important information on pore and stomatal biology. A critical limitation to methods used to observe plant-microorganism interactions in the pore is the application of too much pressure to the sample during observations and z-stack acquisitions. To solve this issue, we recommend the use of a long working-distance water immersion objective lens that allows observations even with thick samples.

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.

Standard optical microscopes are based on diffraction imaging, and their resolution is limited to half the wavelength of the illuminating light. In contrast, scattered-light imaging depends on the sharpness of the optics and is not limited by the wavelength. This technique is used mainly for reflective dark-field observations. The combination of a scattered-light illumination system and sharp optics may improve the resolution of the transmitting microscope. It is well known that the large objective lens in a telescope improves the resolution by reducing the diffraction rings. The digital microscope, designed for industrial applications, has a large objective lens and a 16-bit high-gradation image-processing system, resulting in a sharper image. In this paper, a digital microscope is compared with a standard microscope using the same condenser lens in both cases, which produces scattered light. To amplify the scattered light, the light beam around the optical axis is masked in the condenser lens. Although the standard microscope uses an objective lens with a higher numerical aperture (NA = 0.92) than the digital microscope (NA = 0.82), the digital microscope achieved a higher resolution (over 2500 line-pairs/mm) than the standard microscope, even under dry conditions. This resolving power is greater than that predicted by classical optical theory.

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.

For most of the microelectronics industry history, optical lithography has been the backbone for continuing the trend of making features even smaller. The intention of immersion lithography is to increase the index of refraction in the space between the lens and wafer by introducing a high refractive index liquid in place of the low refractive index air that currently fills the gap. Because the liquid acts as a lens component during scan-step process, it must maintain a high and uniform optical quality. Thus, an immersion unit structure must be implemented to keep the flow field from leaking. After analyzing the mechanics of the flow-field in immersion lithography, an immersion unit structure with double gas-curtain sealing and gas-fluid mixing phase collecting was designed and implemented, featuring chemical surface characteristics. Experimental results were analyzed in terms of vacuum degree in collection antrum, input pressure for gas sealing, bubble-trap within flow field, double gas-curtain, double mixing-phase collection, surface characteristics of wafer.

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.

A microlens of numerical aperture (NA) 0.85 a small-form-factor optical pickup, following the specifications of the Blu-ray disc (BD), was designed, fabricated and evaluated. To avoid difficulties in the fabrication of a high-NA objective lens and to obtain a low chromatic aberration, a new hybrid lens unit was designed to have a refractive lens and a diffractive lens. The micro-plano-aspheric refractive lens was fabricated using glass molding technology, and the diffractive lens was fabricated in a two-dimensional array using the electron beam mastering and consecutive UV embossing process. For the evaluation of the developed lens unit, diffraction efficiency was measured with the proposed diffraction efficiency measurement method, and the wavefront error of the lens unit was evaluated using a modified Mach–Zehnder interferometer. The measured average efficiency of the diffractive lens was approximately 85% and the RMS wavefront error of the lens unit was 0.0376 λrms.

In scanning microscopy by total internal reflection with thermal lens spectroscopy, its spatial resolution depends on the distance between the sample and a converging lens, which corresponds to the objective lens in an ordinary optical microscope. It was found that the resolution was best when the signal induced by the thermal lens effect was maximum. The distance was precisely adjusted by monitoring the signal intensity, and the resolution became twice better than that previously reported. Using a shorter focal-length lens, a resolution of 1.9 microm was attained.

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.

1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI C18(3), hereinafter called “DiI”) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiI C18(5), hereinafter called “DiD”) are fluorescent lipid analogs used to stain artificial and biological membranes. These dyes are widely used in living cell imaging studies (e.g., Danvey et al. 2008; Li et al. 2008; Leung 2009). In confocal laser scanning microscopy, DiI is usually excited by a 543- or 561-nm laser line and yields emission in the 575- to 632-nm range, whereas DiD is excited by a 633-nm laser line and emits in the 650- to 720-nm range (data according to Invitrogen; Carlsbad, CA). Both dyes consist of two indole groups linked by a polymethine chain containing three or five carbon atoms for DiI and DiD, respectively. Membrane affinity of these dyes is conferred by the presence of two long hydrocarbon chains. There are also available derivates that are compatible with long-term preservation after tissue fixation. Recently, we made an interesting observation: prolonged laser illumination of DiD-stained (Vybrant DiD cell-labeling solution, product no. V22887, lot 550865; Invitrogen) living erythrocytes or HeLa cells by dedicated wavelength light during time-lapse confocal imaging causes the appearance of a new emission peak at shorter wavelengths while the original emission peak is reduced (Fig. 1A,​,B).B). To excite DiD, we used a 633-nm laser line (10 µW). Simultaneously, the specimen was illuminated with a 561-nm laser line (5 µW) and a 488-nm laser line (8 µW). For imaging, we used an LSM 510 META microscope with a 40× C-Apochromat NA 1.2 water immersion objective (Zeiss; Oberkochen, Germany). We used the dichroic mirror NT80/20, which does not affect the shape of dye emission spectra. The pinhole was set to 80 µm (~1 AU). The fluorescence signal was directed to the spectral META detector (Zeiss), which was set to collect fluorescence from 490 to 740 nm. The gain of the META detector (Zeiss) was set to 757 for erythrocyte and 719 for HeLa cell imaging. At the beginning of imaging of either stained erythrocytes or HeLa cells, we observed only a single emission peak centered at about 670 nm according to the specification provided by the manufacturer. After about 1 min of continuous imaging, a new peak appeared at shorter wavelengths centered at about 570 nm (Fig. 1A,​,BB and Suppl. Fig. S1A,B). Figure 1. Changes in emission spectra of DiI C18(3) (DiI) and DiI C18(5) (DiD) in membranes of erythrocytes and HeLa cells. (A) Emission spectra of DiD obtained from stained erythrocytes. Imaging details are described in the text. Erythrocytes (16 × 106) ... A similar effect was observed for DiI (Vybrant DiI cell-labeling solution, product no. V22885, lot 575665; Invitrogen). DiI-stained erythrocytes or HeLa cells were excited with a 561-nm laser line (100 µW), and fluorescence was collected by using a 40× C-Apochromat NA 1.2 water immersion objective (Zeiss) and was directed into the detector through an LP 575 emission filter (channel LP575). The cells were also sequentially illuminated with a 488-nm laser line (8 µW) and fluorescence collected by a second detector dedicated for narrow eGFP-like (enhanced Green Fluorescent Protein) emission through a BP505–530 filter (channel BP505–530). Pinholes in both channels were set to 1 AU, and detector gain for erythrocyte imaging was set to 879, whereas for HeLa cells, imaging was set to 750. DiI fluorescence after excitation with a 488-nm laser line can be detected in the channel of BP505–530 nm; however, in our experiments, the detector gain and the laser power were adjusted so at the beginning of the experiments, the signal in the eGFP-dedicated channel was kept slightly above detection threshold (Fig. 1, upper row in C and D). However, after acquiring several frames, a signal in the channel of BP505–530 nm was much stronger than at the beginning of imaging while the original emission signal was reduced (Fig. 1, lower row in C and D, and Suppl. Fig. S1C,D). In the case of DiI, we were unable to detect any changes in emission spectra of the dye by using the META detector (Zeiss) presumably due to lower sensitivity of this detector. Unstained cells imaged in the same conditions did not reveal even weak autofluorescence in any of the investigated spectral regions. To the best of our knowledge, this is the first report that shows the changes of fluorescence spectra of DiI and DiD employed to stain living cells, which were evoked by laser illumination. The described phenomena could strongly affect fluorescence resonance energy transfer measurements with an acceptor bleaching technique in a similar fashion to that previously reported for the CFP (Cyan Flourescent Protein) and YFP (Yellow Flourescent Protein) pair (Kirber et al. 2007). It could also lead to other artifacts such as impression of the probe relocalization in multiple labeled imaging of living specimens. The exact mechanism of the described phenomena remains unknown to us. There is a possibility that upon illumination, the molecules of the dyes undergo either chemical reaction or aggregation that leads to the formation of new species of the dyes with different fluorescent properties. Further studies are required to elucidate the molecular basis of the observed phenomena.

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.

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.

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.

In recent decades, enormous strides have been made in non-invasive imaging of deep tissues and viscera with the development and refinement of ultrasonography, computerized axial tomography, magnetic resonance imaging, and positron emission tomography, to name a few. Progress in non-invasive skin imaging, however, has been slower than in other specialties due in part to the ease with which skin is visually examined and biopsied. The pace of progress in skin imaging, however, has picked up significantly during the past decade. Digital skin imaging, standardized wholebody photography, and dermoscopy are now being used routinely in many centers to help guide the decision to biopsy pigmented skin lesions (Marghoob et al, 2003). More recently, high-resolution optical imaging technologies previously confined to the bench top are finding their way to the bedside (Gonzalez et al, 2003). In this issue of the Journal of Investigative Dermatology, an article from the Medical University of Graz, Austria speaks to the potential utility of reflectance confocal scanning laser microscopy (reflectanceCSLM) for the non-invasive bedside evaluation of pigmented lesions. This study and others like it augur a sea change in the way we will view skin processes clinically at the bedside and in small animal translational research studies in the not too distant future. Among the available non-invasive techniques for skin imaging in vivo, confocal microscopy has been the first to deliver single-cell resolution. A currently available commercial reflectance-CSLM utilizes a diode laser, at the nearinfrared wavelength of 830 nm and a 30, 0.9 numerical aperture water immersion objective lens, to illuminate skin with a low power of 2–10 mW, and visualize a microscopic ‘‘optical section’’ within the superficial layers of the skin (http://www.lucid-tech.com). Nuclear and cellular detail within the epidermis, and microcirculation and collagen with the dermis are imaged in en face sections (oriented parallel to the skin surface) with a measured lateral resolution of 0.7 mm and an axial resolution (i.e., optical section thickness) of 3 mm, to a maximum depth of 100–200 mm (depth of the papillary dermis and superficial reticular dermis) (Rajadhyaksha et al, 1995, 1999). Note that the confocal resolution is similar to that of histology; in particular, the confocal section thickness is comparable to the typically 5 mm thin sections that are prepared for routine histology. Back-scattering of the incident laser light due to variations in the refractive indices of organelles and ultrastructures within the tissue provides contrast in the confocal image (Rajadhyaksha et al, 2004). At a wavelength of 830 nm, melanosomes—due to their size being on the same order as the illumination wavelength, and melanins—due to their high refractive index relative to that of the surrounding skin, produce strong back-scatter. Therefore, melanocytes and basal keratinocytes rich in melanosomes and melanin appear bright and with high contrast in reflectance confocal images (Rajadhyaksha et al, 1995, 1999, 2004). In this issue of the Journal of Investigative Dermatology, Gerger et al (2004) describe excellent sensitivity and specificity achieved for the diagnosis of melanoma using reflectance-CSLM, based on morphologic features that were defined a priori from earlier studies (Langley et al, 2001). In a series of 117 melanocytic skin tumors, consisting of 90 benign and 27 malignant (five in situ, 22 invasive) melanomas, four of the five observers had diagnostic sensitivities and specificities ranging from 93% to 100%. Statistical analysis showed excellent to perfect intra-observer and inter-observer agreement among all five observers for the ten confocal morphologic attributes studied. Of note, none of the five observers (which included two dermatology residents, one dermatologist, and two dermatopathologists) had prior experience in the interpretation of reflectance confocal images. They received a 30 min presentation that instructed them in the confocal morphologic features of benign nevi versus melanomas, using six images of each. None of the images in the instruction set were used in the analysis, and the diagnoses were subsequently made strictly on the basis of the images without knowledge of the clinical setting, clinical appearance, or dermoscopic appearance of the lesions. The authors point out the limitations of their study, which include the pre-selection of the confocal images by a single observer who was not blinded to the clinical/dermoscopic appearance of the lesions and the limited generalizability of the results, given the number of lesions and observers in the data set. A further critique of the study stems from the lack of histology for a subset of the benign melanocytic tumors. Nonetheless, the results provide a ringing endorsement for further assessment of the utility of reflectance-CSLM for the bedside quasi-histologic evaluation of pigmented lesions (Pellacani et al, 2004). Right now, reflectance-CSLM is too expensive and laborious to be considered as a screening tool for melanoma, even if the excellent sensitivity and specificity data presented by Gerger et al (2004) stand up to further scrutiny. With minor advances, however, this technology may become practical for assessing pigmented lesions that have been selected as suspicious on the basis of clinical Abbreviations: reflectance-CSLM, reflectance confocal scanning laser microscopy

Quantitative phase imaging has many applications for label-free studies of the nanoscale structure and dynamics of cells and tissues. It has been demonstrated that optical coherence phase microscopy (OCPM) can provide quantitative phase information with very high sensitivity. The excellent phase stability of OCPM is obtained by use of a reflection from the microscope cover glass as a local reference field. For detailed intracellular studies a large numerical aperture (N.A.) objective is needed in order to obtain the required resolution. Unfortunately, this also means that the depth of field becomes too small to obtain sufficient power from the cover glass when the beam is focused into the sample. To address this issue, we designed a setup with a dual-beam sample arm. One beam with a large diameter (filling the 1.2 N.A. water immersion objective) enabled high-resolution imaging. A second beam with a small diameter (underfilling the same objective) had a larger depth of field and could detect the cover glass used as a local phase reference. The phase stability of the setup was quantified by monitoring the front and back of a cover glass. The standard deviation of the phase difference was 0.021 rad, corresponding to an optical path displacement of 0.9 nm. The lateral and axial dimensions of the confocal point spread function were 0.42 and 0.84 μm, respectively. This makes our dual-beam setup ideal for three-dimensional intracellular phase imaging.

With the purpose of designing a variable-focal lens with large optical aperture, a liquid lens with liquid-membrane-liquid structure and 30mm optical aperture is proposed. Function of the inserted membrane is stated that much stronger elastic force takes place of interface tension, and enlarging aperture size of liquid lens becomes possible. Mechanics analysis of membrane’s deformation and finite element simulation was employed to demonstrate the elastic force maintains the deformation into a parabolic shape. Moreover, a prototype lens was designed and optical performance with a refractive power range of 7.7Diopters, and 7.13line-pair/mm resolution was measured in experiments.

Conventional lens stops, implemented with an absorptive physical aperture, have an angle-dependent projection that introduces field dependent loss and reduces diffraction-limited resolution. Retro-telephoto lenses obtain uniform response using aberration vignetting, but this results in low wide-angle resolution and significant lens volume. However, an angle-independent "virtual" aperture can be created by total internal reflection (TIR) from a thin low index layer inside the lens. We apply this to monocentric wide-angle imaging and find a simple relationship between the filtering layer index and radius and the resulting lens F/#. We provide two detailed designs of lenses with 12 mm focal length and a F/2.5 TIR stop, one using a low index adhesive within a solid fixed-focus lens, the other using an air cavity within an adjustable focus lens. We show the designs provide absolutely uniform resolution and light collection over an angle range of 84° and 106°, respectively, resulting in a dramatic improvement of both light collection and angular resolution per unit volume over conventional wide-angle lenses.

We consider the axial resolution of confocal optical systems employing high numerical aperture objective lenses for the cases when the specimen refractive index is both higher and lower than that of the immersion medium. In the latter case a spherical aberration-like phase shift is introduced when the objective lens numerical aperture is greater than the refractive index of the specimen which leads, inter alia, to an asymmetrical axial response with higher side lobe levels. Theoretical and experimental results are presented.