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Laser scanning confocal microscopy is the workhorse epifluorescence imaging technique used in laboratories worldwide to acquire three-dimensional images of both fixed and live specimens with fine, high-contrast optical sections to discern details that cannot be afforded by standard widefield microscopy. This basic protocol steps the user through a typical three-color imaging experiment using a Zeiss LSM 880 confocal microscope for the example. The extensive Notes section attempts to generalize the method so that concepts and considerations can be applied to other laser scanning confocal systems.

Significance: Differential phase contrast (DPC) is a well-known imaging technique for phase imaging. However, simultaneously acquiring multidepth DPC images is a non-trivial task. We propose simultaneous multiplane DPC imaging using volume holographic microscopy (VHM). Aim: To design and implement a new configuration of DPC-VHM for multiplane imaging. Approach: The angularly multiplexed volume holographic gratings (AMVHGs) and the wavelength-coded volume holographic gratings (WC-VHGs) are used for this purpose. To obtain asymmetric illumination for DPC images, a dynamic illumination system is designed by modifying the regular Köhler illumination using a thin film transistor panel (TFT-panel). Results: Multidepth DPC images of standard resolution chart and biosamples were used to compare imaging performance with the corresponding bright-field images. An average contrast enhancement of around three times is observed for target resolution chart by DPC-VHM. Imaging performance of our system is studied by modulation transfer function analysis, which suggests that DPC-VHM not only suppresses the DC component but also enhances high-frequency information. Conclusions: Proposed DPC-VHM can acquire multidepth-resolved DPC images without axial scanning. The illumination part of the system is adjustable so that the system can be adapted to bright-field mode, phase contrast mode, and DPC mode by controlling the pattern on the TFT-panel.

The light (or optical) microscope is the icon of science. The aphorism "seeing is believing" is often quoted in scientific papers involving microscopy. Unlike many scientific instruments, the light microscope will deliver an image however badly it is set up. Fluorescence microscopy is a widely used research tool across all disciplines of biological and biomedical science. Most universities and research institutions have microscopes, including confocal microscopes. This introductory paper in a series detailing advanced light microscopy techniques explains the foundations of both electron and light microscopy for biologists and life scientists working with the mouse. An explanation is given of how an image is formed. A description is given of how to set up a light microscope, whether it be a brightfield light microscope on the laboratory bench, a widefield fluorescence microscope, or a confocal microscope. These explanations are accompanied by operational protocols. A full explanation on how to set up and adjust a microscope according to the principles of Köhler illumination is given. The importance of Nyquist sampling is discussed. Guidelines are given on how to choose the best microscope to image the particular sample or slide preparation that you are working with. These are the basic principles of microscopy that a researcher must have an understanding of when operating core bioimaging facility instruments, in order to collect high-quality images. © 2020 The Authors. Basic Protocol 1: Setting up Köhler illumination for a brightfield microscope Basic Protocol 2: Aligning the fluorescence bulb and setting up Köhler illumination for a widefield fluorescence microscope Basic Protocol 3: Generic protocol for operating a confocal microscope.

International audience; Interference microscopy is a non-destructive full-field imaging method, mainly used to measure the surface topography of different samples. In this work, two designs for improving the signal quality are described. The first consists of an original vertically orientated breadboard interferometer, in a Linnik configuration. The mechanical design of the arms allows the independent control and alignment of the coherence and the focal plane positions for optimizing fringe contrast. A low noise 16-bit camera is used to improve the sensitivity. The second interferometer is based on a Thorlabs tube system, with a Nikon Mirau Objective and a white LED, all controlled with IGOR Pro software or Labview, with the aim of being more compact, flexible and mobile. For both systems, an evaluation of the interferometric signal quality is performed, whereas the difference in lateral resolution by considering the 3D nature of the interferometric system, or only its 2D imaging abilities, is explored.

In this paper, the three-dimensional measurement technology of structured light is combined with high-speed photography to measure and reconstruct the surface deformation of aluminum alloy target under the condition of light gas gun loading. The test designed a structured light projection system based on the Kohler illumination structure, and adopted a self-developed high-speed photoelectric camera to receive the deformed fringe image. A 100ns magnitude time-resolved three-dimensional surface measurement of the deformation of the target during the process of the light gas gun pushing the projectile into the target plate is realized. By comparing the results of 3D surface reconstruction with numerical simulation results, the reliability of the proposed method for 3D measurement under high speed impact conditions is verified.

Microscopes with an adjustable condenser can be aligned or set-up for Kohler illumination. Detailed instructions, as well as a short guide, facilitate the correct alignment of the microscope in order to maintain a uniformly illuminated high power field.

RESUMEN: La espectroscopia de ruptura inducida por láser (LIBS) es un tipo de espectroscopia de emisión atómica que permite obtener información sobre la composición de materiales o la presencia de determinadas sustancias mediante el análisis de la luz del plasma generado al hacer incidir un láser multipulso de alta energía sobre la superficie de un material en cualquier estado de agregación. La captura de luz se puede realizar a través de varios métodos: fibra óptica, imagen por lente, telescopio remoto... Estas formas de captura cuentan con numerosas ventajas e inconvenientes, siendo los principales problemas la pérdida de luz y las fluctuaciones del plasma con el tiempo, lo cual genera resultados diferentes para cada medición. En este trabajo se van a intentar solventar dichos problemas buscando un sistema de lentes que forme imágenes homogéneas en el tiempo y que capte la mayor cantidad de luz posible. Para ello, se utilizará el software de diseño óptico OpticStudio de Zemax, con el que se realizarán las simulaciones del comportamiento de la emisión del plasma a su paso por los diferentes sistemas ópticos, y, posteriormente, se comprobará la eficacia de dichas simulaciones experimentalmente. ABSTRACT: Laser-Induced Breakdown Spectroscopy (LIBS) is a type of atomic emission spectroscopy that allows to obtain information on the composition of materials or the presence of certain substances by analyzing the light from the plasma generated when a high-energy multipulse laser strikes on the surface of a material in any state of aggregation. Light capture can be done through various methods: fiber optics, lens imaging, remote telescope... These forms of capture have numerous advantages and disadvantages, the main problems being the loss of light and plasma fluctuations over time, which produces different results for each measurement. The objective of this dissertation is to solve these problems by looking for a lens system that forms homogeneous images over time and captures as much light as possible. For this purpose, the OpticStudio software by Zemax will be used to simulate the behavior of the plasma emission as it passes through the different optical systems, and, subsequently, the effectiveness of these simulations will be experimentally verified. Grado en Física

The problem that is posed byphase-only" objects, such as epithelial cells, for brightfield microscopy has resulted in development of several specialized imaging techniques including phase-contrast and DIC. In the past decades there has been increasing research on quantitative phase imaging (QPI), which enables real-time cellular dynamics to be visualised and the 3-D morphology to be quantitatively measured. It has previously been demonstrated that the DIC and phase-contrast images can be computationally generated using the phase-image provided by QPI. Recently, we have extended this approach to include Rheinberg. Although not as popular as phase contrast or DIC, Rheinberg illumination provides a form of label-free optical staining by introducing a multi-color filter into the condenser plane of the microscope, enabling different features within the cell to be stained with different colors depending on their spatial-frequency content. We recently developed a theory for image formation with Rheinberg illumination under the conditions of Kohler illumination from which an algorithm was developed that could simulate this process using the QPI image as input. In this paper we review and further develop this approach by testing it with multiple different modalities for recording the QPI image, namely digital holographic microscopy, which uses coherent illumination and spatial light interference microscopy, which makes use of white light. We examine a variety of samples including diatom and epithelial cells using a number of microscope objectives with different numerical apertures.

Low level light therapy (LLLT) is a fast-growing technology used in noninvasive therapies which can stimulate healing and relieve pain and inflammation. The introduction of light-emitting diode (LED) devices has reduced many of the concerns associated with halogen lamp and lasers, such as expense and safety. However, many LED devices are bulky and not designed for home use. Besides, the effectiveness of the treatment by LLLT has significant variations in terms of dosimetry parameters for the used LED, such as wavelength, irradiance or power density, energy, etc. Therefore, we design a home-used multi-wavelength LED phototherapy system with the advantage of a small volume and high irradiance in this paper. By using AlN ceramic substrate and copper substrate as composite substrate, the vertical structure chip is tightly attached to the ceramic substrate circuit, then the package of the four-wavelength chip module is completed. In order to distribute the irradiance of all four different spectra over the desired target area, an optical mixing rod is used to collect the light, and the light is all reflected into the output face of the optical mixing rod through full internal reflection. Additionally, to form a light spot of 50mm in diameter at a distance of 5cm from the output surface, we design a pair of lens with a short focal length and large field of view based on Kohler illumination method. The parameters of the lens are designed by the software of ZEMAX. The hardware circuit uses the STM32F104 processor as controller and constant current drive mode to ensure the quality of LED light. An experimental prototype is established, and the irradiance and its uniformity, stability and safety of the phototherapy system are tested. The experimental results show that the maximum irradiance of the system is 115.4mW/cm2, and the uniformity reaches 88%. After the phototherapy system works for 2 hours with maximum illumination, the irradiance deviation is 6.7% and the temperature rise of the system is 19°C.

Magneto-optical Kerr effect (MOKE) is an efficient approach to probe surface magnetization in thin film samples. Here we present a wide-field MOKE technique that adopts a Köhler illumination scheme to characterize the current-induced damping-like spin-orbit torque (DL-SOT) in micron-sized and unpatterned magnetic heterostructures with perpendicular magnetic anisotropy. Through a current-induced hysteresis loop shift analysis, we quantify the DL-SOT efficiency of a Ta-based heterostructure with bar-shaped geometry, Hall-cross geometry, and unpatterned geometry to be |ξ DL | ≈ 0.08. The proposed wide-field MOKE approach therefore provides an instant and direct characterization of DL-SOT, without the need of any further interpretation on electrical signals.

Every veterinary practice should be equipped with a microscope and all veterinary nurses should become competent with its use. A basic knowledge of the precise setup of the microscope is essential for image quality and prompt results.

An active illumination microscope provides simultaneous acquisition of multi-contrast images such as bright-field, dark-field, and differential phase-contrast using various illumination patterns. We experimentally demonstrate the multi-contrast images of structural modifications produced in glass by a femtosecond laser. The multi-contrast images of structural modifications were obtained by changing illumination patterns with a projector for microscopic contrast enhancement. The refractive index change and scattering damage produced in BK7 glass were identified by differential phase-contrast images and dark-field images. We can enhance the direction of contrast in the imaging of modifications by changing the rectangular illumination pattern. Multi-contrast microscopy by different illumination pattern is a technique for in situ monitoring of modifications by femtosecond laser pulses in transparent materials.

A compact microlens array (MLA) integral homogenizer composed of a projection MLA, a condenser MLA, and a subimage array mask based on Kohler illumination is presented herein. By adopting the optimal design of an aspheric projection sublens, a short-distance integrator for unconventional geometric irradiance tailoring can be acquired. Compared with the traditional integrator, the integral lens is removed in the proposed integrator. An incident beam with a larger divergence angle is permitted while a sharp illumination performance is maintained. In addition, the illumination distribution with a predefined geometrical profile can be obtained by introducing a subimage array mask without replacing the MLA elements. Through the introduced mask, the illumination attenuation area is cut, the distortion is caused by the large NA, and the peak effect caused by the integral lens of the traditional integrator can be eliminated. The subimage array mask is generated by combining ray tracing and the radial basis function image warping method. By introducing the array mask into the system, an 80% edge relative illumination and an 85% overall illumination uniformity are realized for a compact large-NA integrator with N A = 0.3 and thickness of 4.5 mm. An 88% edge relative illumination and a 92% overall illumination uniformity are realized for a small-NA integrator with N A = 0.15 .

Wide-field saturated structured illumination microscopy has not been widely used due to the requirement of high laser power. We propose a novel method called laser scanning saturated structured illumination microscopy (LS-SSIM), which introduces high order of harmonics frequency and greatly reduces the required laser power for SSIM imaging. To accomplish that, an excitation PSF with two peaks is generated and scanned along different directions on the sample. Raw images are recorded cumulatively by a CCD detector and then reconstructed to form a high-resolution image with extended optical transfer function (OTF). Our theoretical analysis and simulation results show that LS-SSIM method reaches a resolution of 0.16 λ, equivalent to 2.7-fold resolution than conventional wide-field microscopy. In addition, LS-SSIM greatly improves the optical sectioning capability of conventional wide-field illumination system by diminishing our-of-focus light. Furthermore, this modality has the advantage of implementation in multi-photon microscopy with point scanning excitation to image samples in greater depths.

For requirements of light source on tiny parts measurement system, this paper puts forward a single wavelength LED to design uniform light of multiple ring. According to detection principle of tiny parts, the design method of ring source is set up, as well as luminescence radiation calculation model of LED ring source. The calculation model of light illumination properties of LED ring array light source are set up and analyzed, light illumination calculation functions of ring source are derived; based on design characteristics of ring source, illumination model and calculation principles of uniform light source are analyzed, the derivation process is established in detail. Through calculation and experimental analysis, illumination uniformity distribution curve of LED ring array light source were given under different detecting plane and the horizontal distance of LED, and the contrast results of illumination uniformity in the x axis and y axis also were given, the results show that when the distances between detecting plane and LED plane are 80 mm and 100 mm, illumination uniformity of LED ring array light source can reach 0.96.

In traditional optical microsopy the uniformity of off-axis illumination is poor. In this paper we propose a novel LED optical system for near-field illumination in microscopy. The system includes a LED light source, an optical collimator and a freeform, micro-reflective lens array. The Monte Carlo method is used to simulate the system. The simulation results show that the illumination uniformity can be better than 85% in the observation zone. The optical system can be put at the side of the main optical axis of the microscope. The illumination uniformity is not varied when the observation distance changes so it has good stablilty.

We present an improved structured illumination configuration for structured illumination microscopy (SIM) based on spatial light modulator. Precise phase shifts and rotation of illumination fringes can be dynamically controlled using a spatial light modulator. The method is different from the conventional illumination configuration that are based on interference of ±1 diffractive order light. The experimental setup requires less optical elements making it compact, reliable, and suitable for integration. The method has been applied in the standing-wave total internal reflection fluorescent microscopy. High lateral resolution of sub-100 nm was achieved in single directional resolution enhancement experiments.

The use of non-invasive hyperspectral imaging techniques has become standard practice in the materials analysis and study of precious cultural heritage objects such as drawings, paintings, murals and more. However, the non-linear mixing of spectral signatures from complex and heterogenous objects with multiple colorants present below the resolution limits of the camera can complicate material identification. Consequently, ground truth measurements are still usually obtained from microscopic samples removed and embedded to expose stratigraphy and obtain sub-surface information about the artist’s material choices and technique. This work considers a microscopic spectral imaging technique capable of mapping molecular information in such micro samples at high spatial and spectral resolution while avoiding some of the challenges of complimentary techniques, such as swamping fluorescence in Raman spectroscopy or long integration times using FT-IR spectroscopy. Construction of a dark field hyperspectral microscope for cultural heritage samples is described using a tunable light source to illuminate the sample monochromatically from the visible to near infrared wavelengths, with the diffusely reflected light collected from the specimen with a long working distance, 20x objective. The illumination and detection arms were decoupled to better focus the power of the tunable light source across the tunable range through Kohler illumination optics. By mounting the optical train on a rotating arm, we can achieve multiple angles of illumination and optimize lighting conditions. The sample is also rotated in order to reconstruct an even distribution of light across the field of view. This multi-axis movement capability also provides exciting opportunities to leverage more than simple spectral information from an image series such as surface topography and differential phase contrast information. The developed microscope was used create a library of spectral signatures for comparison to painting cross sections, and the ability of the microscope to identify and examine individual pigment particles was tested.

Current LED based structured illumination projectors use commercial objective lenses to project the pattern, leading to an increased size of the projector. We present a compact, pseudo-random pattern generator with an enhanced field of view (FOV) of 60° × 60° to project structured light for machine vision applications. The miniaturization of optics is achieved by utilizing the multi-aperture approach. Currently such multi-aperture projection optics have an FOV of about 20°. Enlarging the FOV of microlenses results in higher geometric aberrations. Aberration correction for wide FOV is done by distributing the optical power over several microoptical surfaces, as in traditional optical systems, leading to a design of stacked microoptics. The correction of dominant aberrations like astigmatism is done by employing anamorphic microlenses. Further enhancement of the FOV is achieved by continuously varying the FOV of the individual lenslets over the microlens array (MLA). Thus, every channel of the array projects light to a different region of the image plane, resulting in a symmetric and continuous filling of the FOV across the image field. Consequently, anamorphic irregular or 'chirped' MLAs with lenslets of varying focal length and orientation depending upon its location in the MLA are designed. This also requires the projection optics to be illuminated with a field dependent incidence for which a two lens condenser optic is designed. A regular condenser lens array couples the light into the channels similar to the scheme for Kohler illumination, while the stacked microoptics along with a bulk field lens image the slide object on to the projection plane. A corresponding prototype with stacked and chirped microoptics with buried apertures and slide structures is realized using advanced reflow technology and characterization of such an arrayed pattern projector is presented.

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

Laser-induced breakdown spectroscopy (LIBS) has recently demonstrated its unrivaled performance for broadband elemental imaging of surfaces. The dimensions of the laser sampling spot still being potentially larger than the interfaces of chemical domains, the plasma created at each location can be largely varying and inhomogeneous with contributions from the different sides of the interface. This variation can become problematic when imaging it on fiber bundles connected to multiple spectrometers. A spatially heterogeneous signal would lead to spatially dependent image on the fiber bundle causing inconsistent readings and loss of efficiency. Köhler illumination is used in this study to create a homogenous illumination, regardless of the source homogeneity, thus improving light collection efficiency. The performance of this approach was demonstrated with inhomogeneous spectral sources and applied to the LIBS analysis of a metallic interface, showing up to a sixfold improvement of the homogeneity of the plasma collection.

In order to meet the increasing demand for the diagnosis of household medical eye fundus, the design of a new fundus camera is proposed. The entire structure includes the imaging system and the lighting system. The optical imaging system, modeled on the Kepler telescope, matches the exit pupil of the human eye and the entrance pupil of the mobile phone camera. Thus, this system uses a mobile camera lens that owns more than 5 million pixels to take a picture of a clear fundus. A new type of fundus camera is designed with the field of 40°, the working distance of 30mm and the full length of 138 mm. The value of MTF outweighs 0.5 at the central field of view. The lighting system takes advantage of the Kohler illumination. The stray light of the reflection of corneal is effectively suppressed by adding an annular aperture on the optical path. The inner diameter of the annular light spot formed at the cornea was larger than 4mm, the outer diameter was less than 6mm, and the diameter of the evenly illuminated fundus area was 12mm.

According to the characteristics of the reflective optical microscope lighting system, an improved Kohler illumination system for the full-field optical coherence tomography system (FFOCT) was designed to realize the illumination of biological samples and living biological tissues. The illumination system differs from the conventional Kohler illumination system. The filament of the halogen lamp is imaged on the back focal plane of the microscope objective, then parallel light is incident on the sample plane. The improved Kohler illumination system uses a halogen lamp as the light source and is divided into two parts: the condenser front and rear groups. The front condenser group uses two double-glued structures, and the rear group uses a double-coupled lens. The optical design software Zemax was used to optimize the design, and the illumination analysis software Tracepro was used to trace the ray and simulate the imaging of the light source in the front focal plane of the microscope objective. The entire improved Kohler illumination optical path has a total length of 594 mm, the diaphragm is 122 mm from the front group of the condenser, 99 mm from the rear group, and the working distance is 292 mm; the luminous efficiency of the receiving surface is as high as 60.38%, and the edge of the light spot is smooth and clear. The illumination system makes full use of the optical power emitted by the light source and facilitates the placement of a device such as a splitting prism between the condenser and the microscope objective, which satisfies the requirement of the entire machine well.

In this chapter I discuss the bright-field microscope, perhaps, along with the telescope, one of the most elegant and important scientific instruments ever invented. I discuss some of the discoveries made with the bright-field microscope. I discuss the components of the bright-field microscope, particularly the objective lenses. I discuss the aberrations that can occur in the objective lenses and the methods used to correct them in order to obtain high-quality objectives. I discuss how to establish Kohler illumination and the optical paths taken by the illuminating rays and the image forming rays in the bright-field microscope set up for Kohler illumination. I discuss the use of the bright-field microscope and how to adjust the field and aperture diaphragms to balance resolution, contrast, and depth of field.

In the frame of the Concentrator Photovoltaic (CVP) technology, to achieve ultra-high (UH) concentration levels is a promising strategy to reduce the cost of the PV electricity, since it takes advantage of the reduction of the solar cell area and also of the physical behavior of the solar cells. This work analyzes a previous optical concentrator design for a module with a geometrical concentration of 2000×, i.e. in the range of the UHCPV (ultra-high CPV), in terms of its geometrical design. This optical design can be called Cassegrain-Koehler, since it uses different optical units based on the basic Cassegrain design for telescopes (with a paraboloid primary mirror and with a hyperboloid secondary mirror) as well as takes advantage of the Koehler illumination technique on the solar cell. In order to optimize this design, the f- number of the primary mirrors is varied while the f-number of the secondary mirrors is maintained constant ‒3.5. This allow the impact of the compactness of this UHCPV design to be determined. The results show an optimum trade-off of optical efficiency and acceptance angle when the f-number of the primary mirrors is around 0.63, which is not the most compact design obtained. However, the irradiance uniformity over the solar cell, evaluated through the peak-to-average ratio (PAR) results lowest in the most compact designs.In the frame of the Concentrator Photovoltaic (CVP) technology, to achieve ultra-high (UH) concentration levels is a promising strategy to reduce the cost of the PV electricity, since it takes advantage of the reduction of the solar cell area and also of the physical behavior of the solar cells. This work analyzes a previous optical concentrator design for a module with a geometrical concentration of 2000×, i.e. in the range of the UHCPV (ultra-high CPV), in terms of its geometrical design. This optical design can be called Cassegrain-Koehler, since it uses different optical units based on the basic Cassegrain design for telescopes (with a paraboloid primary mirror and with a hyperboloid secondary mirror) as well as takes advantage of the Koehler illumination technique on the solar cell. In order to optimize this design, the f- number of the primary mirrors is varied while the f-number of the secondary mirrors is maintained constant ‒3.5. This allow the impact of the compactness of this UHCPV design to be...

A two-lens optical system entails two beams: a crossover beam and a Kohler illumination beam. We observed that upon varying the excitation corresponding to the first lens from large to small values, the crossover beam changed to a Kohler illumination beam with increased brightness. We derived new calculation formulas for the Kohler illumination beam. Brightness B and beam current Ib can be expressed as B = Bco (ϕco/ϕ)2 and Ib = Bco (παϕco)2/4, where Bco, ϕco, ϕ, and α denote the crossover beam brightness, crossover beam size, Kohler illumination beam size, and beam semi-angle, respectively; additionally, ϕco and ϕ are obtained at the same first lens excitation. These two equations were experimentally validated. We obtained a brightness of 2.05 × 108 A/cm2 sr using a beam energy and an emission current of 20 keV and 54 µA, respectively. Notably, this value surpasses the Langmuir limit of 4.11 × 105 A/cm2 sr by 499 times.

We propose a new algorithm for recovering both complex field (phase and amplitude) and source distribution (illumination spatial coherence) from a stack of intensity images captured through focus. The joint recovery is formulated as a nonlinear least-square-error optimization problem, which is solved iteratively by a modified Gauss–Newton method. We derive the gradient and Hessian of the cost function and show that our second-order optimization approach outperforms previously proposed phase retrieval algorithms, for datasets taken with both coherent and partially coherent illumination. The method is validated experimentally in a commercial microscope with both Kohler illumination and a programmable light-emitting diode dome.

A 1:1 lithography projection has been designed and is fabricated for a 3D integrated circuit fabrication platform. Using a dual triplet as an initial type to form a one-to-one lens and applying a tele-centric structure, the optical common components of an optical system have been designed. The tolerance of the mechanical mounts is simulated by tilting the mounts to single and two aspheric surfaces of lens to show the degradation in the modulation transfer function; thus, the single aspheric-tilted mount in a system is suggested to reach the precision. Furthermore, Koehler illumination is used. By applying partial coherence analysis, the optimized relative numerical aperture was found. As the system is built, optimized performance should be expected.

To meet the uniform dose exposure in optical lithography, it is desirable to get uniform illumination in the scanning direction on wafer for the step-and-scan lithographic system. We present a flat Gauss illumination for the step-and-scan lithographic system in this paper. Through flat Gauss illumination in scanning direction, pulse quantization effect could be reduced effectively. Correspondingly, the uniformity of the reticle and wafer is improved. Compared with the trapezoid illumination, flat Gauss illumination could keep the slit edge fixed, and pulse quantization effect will not be enhanced. Moreover flat Gauss illumination could be obtained directly without defocusing and blocking, which results in high energy efficiency and high throughput of the lithography. A design strategy for flat Gauss illumination is also proposed which offers high uniformity illumination, fixed slope and integral energy of flat Gauss illumination in different coherence factors. The strategy describes a light uniform device which contains first microlens array, second microlens array, one-dimensional Gauss diffuser and a Fourier lens. The device produces flat Gauss illumination directly at the scanning slit. The design and simulation results show that the uniformity of flat Gauss illumination in two directions satisfy the requirements of lithographic illumination system and the slope. In addition, slit edge of flat Gauss illumination does not change.

In many illumination problems, the beam pattern needed and/or some geometrical constraints lead to very asymmetric design conditions. These asymmetries have been solved in the past by means of arrangements of rotationally symmetric or linear lamps aimed in different directions whose patterns overlap to provide the asymmetric prescriptions or by splitting one single lamp into several sections, each one providing a part of the pattern. The development of new design methods yielding smooth continuous free-form optical surfaces to solve these challenging design problems, combined with the proper CAD modeling tools plus the development of multiple axes diamond turn machines, give birth to a new generation of optics. These are able to offer the performance and other advanced features, such as efficiency, compactness, or aesthetical advantages, and can be manufactured at low cost by injection molding. This paper presents two examples of devices with free-form optical surfaces, a camera flash, and a car headlamp.

In recent years, high-resolution microscopy using structured illumination has been practically applied for fluorescent bio-imaging. However, there is a large amount of speckle noise in reflected- and scattered-light images, because structured illumination is typically generated by laser-beam interference. Hence, this high-resolution imaging technique cannot be effectively used in industrial applications. In this study, we attempted to generate structured illumination using two-beam interference of low-coherence light for high-resolution and low-speckle imaging. First, we constructed an optical system consisting of a Michelson interferometer configured in such a manner that it achieved zero optical path-length difference and allowed the interference fringes to be manipulated. Then, we confirmed that the generated structured illumination width corresponded to the coherence length of the light source. As a final result of the resolution improvement experiment, the narrow sample pitch of 0.4 μm was successfully resolved beyond the diffraction limit of 0.74 μm with relatively less speckle noise.

Recent advances in survey design have led to conventional common-midpoint-based analysis being replaced by subsurface-based seismic acquisition analysis, with emphasis on advanced techniques of illumination analysis. Among them is the so-called focal beam method, which is a wave-equation-based seismic illumination analysis method. The objective of the focal beam method is to provide a quantitative insight into the combined influence of acquisition geometry, overburden structure, and migration operators on the resolution and angle-dependent amplitude fidelity of the image. The method distinguishes between illumination and sensing capability of a particular acquisition geometry by computing the focal source beam and the focal detector beam, respectively. Sensing is related to the detection properties of a detector configuration, whereas illumination is related to the emission properties of a source configuration. The focal source beam analyses the incident wavefield at a specific subsurface grid point from all available sources, whereas the focal detector beam analyses the sensing wavefield reaching at the detector locations from the same subsurface grid point. In the past, this method could only address illumination by primary reflections. In this paper, we will extend the concept of the focal beam method to incorporate the illumination due to the surface and internal multiples. This in fact complies with the trend of including multiples in the imaging process. Multiple reflections can illuminate a target location from other angles compared with primary reflections, resulting in a higher resolution and an improved illumination. We demonstrate how an acquisition-related footprint can be corrected using both the surface and the internal multiples.

High-resolution light distributions are lately in demand for vehicle headlamp systems as an innovative lighting approach. This lighting approach can realize functionalities, such as precise glare avoidance and on-road projection, which are useful for improving traffic comfort and safety. For achieving the required high-resolution light distribution, area-based projection technologies, such as DMD, LCD, and LCoS, are considered to be integrated into such headlamps. These projection devices demand rectangular illumination areas with specific light distributions to fulfill the requirements for illumination efficiency and performance in headlamp systems. Lenslet arrays, based on the principle of K&ouml, hler illumination, can effectively homogenize the light and shape it into rectangular shapes simultaneously. Such components are widely used in projection applications. However, they also show functional potentialities to be applied in high-resolution headlamps. This paper explains the design principles and methods of lenslet arrays for beam pre-shaping in headlamp systems. It validates the homogenization using a self-designed and manufactured lenslet array in a demonstrator in the first place. Afterward, this paper introduces two new methods for the centralized beam shaping required by some headlamps. These methods are validated by optical simulations.

High efficiency coupling of the array of UV light diode light source could be able to replace the traditional mercury light source, while mercury light source requires a large amount of electricity, condenser optics and volume, with hazard environmental issue. It is an urgent need for further requirement of light source for lithography. Although array UVLED could be a replacement of light sources of lithography, there are some existent optical parameters that not fit for optical coupling, such as large divergent cone angle of single chip and half divergent angle of array, which are weak the possibility of application in lithography. Before the broadly accepted by lithography, those shortages have to be overcome. Some techniques, such as designing small divergent cone angle of single chip, or eliminating divergent angle have suggested by fly eye and other methods, yet all of those methods, only improve the coupling efficiency and uniformity of emitted surface in some limited extent. The project is to develop a high coupling efficiency of the optical system used in the array of UV light diodes, which can make the high coupling efficiency, despite UVLED has a small size and large divergent angle. The optical design of illumination has carried out for array UVLED, an illumination optics has designed based on Koehler type illumination, and it has shown reducing the divergent angle to increase in coupling efficiency between the Array UVLED to the mask of lithography. The uniformity of average power has shown excellently fitted for high quality lithography in direct exposure or lens exposure.

Accurate optics-based dimensional measurements of features sized well-below the diffraction limit require a thorough understanding of the illumination within the optical column and of the three-dimensional scattered fields that contain the information required for quantitative metrology. Scatterfield microscopy can pair simulations with angle-resolved tool characterization to improve agreement between the experiment and calculated libraries, yielding sub-nanometer parametric uncertainties. Optimized angle-resolved illumination requires bi-telecentric optics in which a telecentric sample plane defined by a Kohler illumination configuration and a telecentric conjugate back focal plane (CBFP) of the objective lens; scanning an aperture or an aperture source at the CBFP allows control of the illumination beam angle at the sample plane with minimal distortion. A bi-telecentric illumination optics have been designed enabling angle-resolved illumination for both aperture and source scanning modes while yielding low distortion and chief ray parallelism. The optimized design features a maximum chief ray angle at the CBFP of 0.002° and maximum wavefront deviations of less than 0.06 λ for angle-resolved illumination beams at the sample plane, holding promise for high quality angle-resolved illumination for improved measurements of deep-subwavelength structures using deep-ultraviolet light.

Current state-of-the-art technology for in-vitro diagnostics employ laboratory tests such as ELISA that consists of a multi-step test procedure and give results in analog format. Results of these tests are interpreted by the color change in a set of diluted samples in a multi-well plate. However, detection of the minute changes in the color poses challenges and can lead to false interpretations. Instead, a technique that allows individual counting of specific binding events would be useful to overcome such challenges. Digital imaging has been applied recently for diagnostics applications. SPR is one of the techniques allowing quantitative measurements. However, the limit of detection in this technique is on the order of nM. The current required detection limit, which is already achieved with the analog techniques, is around pM. Optical techniques that are simple to implement and can offer better sensitivities have great potential to be used in medical diagnostics. Interference Microscopy is one of the tools that have been investigated over years in optics field. More of the studies have been performed in confocal geometry and each individual nanoparticle was observed separately. Here, we achieve wide-field imaging of individual nanoparticles in a large field-of-view (~166 μm × 250 μm) on a micro-array based sensor chip in fraction of a second. We tested the sensitivity of our technique on dielectric nanoparticles because they exhibit optical properties similar to viruses and cells. We can detect non-resonant dielectric polystyrene nanoparticles of 100 nm. Moreover, we perform post-processing applications to further enhance visibility.

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three-dimensional (3D) samples that allows the use of two-dimensional (2D) data processing. Indeed, obtaining super-resolution images of thick samples is a difficult task if low spatial frequencies are present in the in-focus section of the sample, as these frequencies have to be distinguished from the out-of-focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high-resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out-of-focus content from the raw images. After this cleaning step, we can obtain super-resolution images of optical sections in thick samples using a two-beam harmonic illumination pattern and a limited number of raw images. This two-step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two-dimensional.

Proper use of the microscope is the prerequisite to all of pathology, from learning to achieve parfocality and Kohler illumination to learning how to sit at the microscope properly and avoid ergonomic injury. Upon arriving in the pathology department, you will most likely be given a microscope of your own. Learning to operate the microscope effectively is the prerequisite to everything else in this book. Parfocality means that if an image is focused at 40×, you should be able to switch to 4× and still be in focus. It is not the same as Kohler illumination. You can achieve true parfocality only on a microscope with two adjustable eyepieces; it is most important on multiheaded microscopes, when the observers at the additional heads need to be in sync with the person controlling the focus. The beginning of a session with multiple users on a multihead microscope should always start with this focusing ritual.

In the modern world optical sensor systems have a vast number of applications. In this research work the system composed of the two cameras and the laser illumination with the 49 lasers is considered. In the previous research work we proposed the general calibration technique for this system. It was shown that the most complicated subtask in this technique is determining the beam directions for the laser illumination calibration because it couldn’t be solved using the known algorithms. The main stages which are required to execute for determining the beam directions for the laser illumination are: tracking of the laser illumination points in the image sequence of the calibration object; calculation of the coordinates in space for the found laser illumination points; constructing the laser beams in space passing close as much as possible to the found points. Within the scope of the research carried out, all the main stages for determining the beam directions for the laser illumination are considered. But much attention in this work is devoted to the third stage. The origin of the laser beam is known since it coincides with the known location of the laser on the laser illumination. Thus the problem is to find the ray passing through the origin and the least divergent in mean square from the found set of points. Also the algorithms for performing each stage are suggested. Particularly, we developed our own algorithms taking into consideration specifics of the available system with the laser illumination, viz. the algorithms for detecting the laser illumination points, the algorithms for constructing the laser beam from the found set of points. The beam directions in space can be determined for each laser of the illumination. These determined directions can be used as the subset of the calibration parameters for the whole system.