If optical methods are long time ago used in metrology, coming of laser sources has improved drastically the impact of optics in metrology. The progressive existence of more and more industrial optoelectronic components on the market is responsible of the actual introduction of optical technics in industrial processings like interferometric control, wide-ranging optical sensors , visual inspection..... Further partial coherence and guiding properties of the light field, non linear optical comportment of the medium offer also interesting metrological applications. The aim of this paper is not to give a full description of all the optical methods used in metrology, but to draw some general specific properties and ideas illustrated by representative applications.

The confocal scanning microscope is a coherent system with significantly improved imaging characteristics. The effective pupil function of such an instrument is a composite one, formed by the convolution of the pupil functions associated with the illuminating and imaging optical systems. Because of this additional degree of freedom, the transfer function of the system can be modified with comparative ease. Confocal imaging will be analysed within a very simple but quantitative physical model. Also, the effect of various pupil combinations on the imaging properties of such systems will be presented with special emphasis on the use of superresolving pupils.

It is well known that the type II scanning optical microscope has important advantages over conventional optical microscopes. These advantages lie primarily in their ability to eliminate out of focus signals so that the image contains information from a thin axial slice. In this paper we quantify this effect for several typical microscope configurations. We have calculated the axial response of the microscope as a function of the numerical aperture and the focal length of each lens in the system. The effect of misalignment of the point detector has been investigated. In addition the effect of feature size has been analysed; we have demonstrated that the depth discrimination differs substantially for a point and large area object. These analytical results have been extended numerically to account for arbitrary size objects. The computations have been performed using both Fresnel diffraction theory and a hybrid approach which combines diffraction theory and ray optics. The latter method greatly reduces computational complexity and the results agree with those obtained using diffraction theory in cases where comparison is applicable.

Scanning confocal microscopy has become an important tool in the field of optical sectioning and 3D-reconstruction of biological objects. Usually these samples are embedded in dispersive media, introducing depth dependent spherical aberration. Depth and lateral resolution are therefore impaired. We discuss this problem theoretically and show experimental results.

It has been recently demonstrated that one of the most important applications of Confocal Scanning Laser Microscopy (CSLM) is fluorescence microscopy. In this paper we discuss the application to this case of a general method suggested by two of the authors (M.B. and E.R.P.) for improving resolution. The method requires that the full image is recorded by means of a suitable array of detectors and that a linear integral equation is solved for determining the object at each step of the scanning procedure. We investigate several integral equations related to 1D, 2D and 3D imaging.

The imaging properties of a model high aperture fluorescence confocal microscope are studied by way of numerical methods. The model consists of an aberration-free high aperture objective illuminated by a monochromatic linear or circular polarized light source, and a detection system equipped with a finite sized pinhole. The computation of the intensity distribution near the confocal plane is based on electromagnetic diffraction theory. We study the 3-D imaging properties of the model by examining the images of three different objects; points, lines and planes. We calculate the resolving power for these objects as a function of their orientation and the size of the detector pinhole. The results indicate the necessity for image analysis schemes to take these effects into account.

This paper presents theoretical and experimental research on super-resolution in optical microscopy. The theoretical analysis is based on partially coherent imaging theory. A source of annular form is used to achieved super-resolving imaging and a significant improvement in the resolving power has been observed experimentally. The imaging of a two-point object and two-bar objects of different spaces and widths are discussed. The displacement of the Gaussian image point and problems associated with this technique are also considered.

A scanning optical system has been developed which can simultaneously and independently measure the differential amplitude and phase of light reflected off an object surface. The central element of such a system is an acousto-optic modulator which splits the incoming light into two beams of different optical frequencies, amplitude modulated in phase quadrature. These are focused with close proximity on the sample surface. The theoretical sensitivity of this type of differential optical system is 1x10^-3 mrad in phase and 1 in 10⁵ in reflectivity variation. In this paper we will look at the first implementation of the technique and practical problems encountered with this system. A second system is presented in which two first order beams from the Bragg cell are used to interrogate the sample. An analysis is made of the interpretation of the detected signals when different types of amplitude modulation is used. This latter system is shown to have an improved differential amplitude response. A number of samples have been looked at to assess the performance of the system. These demonstrate the high sensitivity of this system and its capability in separating amplitude and phase information. The advantages of extracting differential amplitude as well as phase information is discussed using a simple layered model. Potential applications include film thickness metrology, surface profilometry and measurement of refractive index variation. In addition it can be used to image low contrast objects such as biological specimens and defects on crystalline structures.

The Coherent Scanning Microscope (CSM) is mainly composed of two objectives arranged in tandem combined with the scanned object. The synchronized scanning of both of the object and the electron beam emitted from the cathod ray monitor will assure the construction of the temporal image. Since it is easy to aligne the first objective of the CSM, while it is not the case for the second objective, hence we assume that the later objective is subjected to tilting and to a lateral shift with respect to the first aligned objective. Another case assumes that the second objective is influenced by coma of third order in addition to the exoentration errors. In both cases, the resulting impulse response h_r= h₁h₂, is calculated. The above mentioned problems of excentration errors combined with wavefront aberration, which are both harmfull for the microscope resolution, are discussed.

The measuring process of laser scanning microscopy is analyzed. The distortion of the recorded image due to finite focus diameter and the temporal response of the electronic recording device is estimated. The influence of noise on the accuracy of the recorded image is considered. Optimum recording conditions such as choice of scan velocity and time constant of the registration device are discussed for minimal total error.

By using a translating phase mask, a Fourier-transforming lens and a spatial filter, we can process laser light reflected from a surface in such a way as to avoid the diffraction effects common in conventional imaging. This optical technique is thus inherently capable of measuring minute surface features such as the width of deposited or inscribed lines with better than Rayleigh resolution and should therefore be applicable for metrology of sub-micron features. The novelty of the technique is to transfer linewidth information into the zero-order spatial frequency component of the light reflected from the surface. We present analyses and computer simulations to detail the effects of such features of the system as the sharpness of a step edge in a phase-shifting mask, the magnitude of the phase shift introduced by the mask, the variation in reflectivity and height of various regions of the surface structure, and the effect of instrumental noise on the determination of linewidth. Experimental measurements were performed on specimens with large feature dimensions to verify the inherent capability of the technique. The results agree well with theoretical predictions. It is hard to validate this technique at smaller dimensions because of the necessity for precise lateral translation of the mask with respect to the surface and the sensitivity of the system to the mask-to-surface distance. We discuss modifications in the next-generation experimental set-up that will address both these issues. Current results indicate that this technique will be viable well into the submicron range.

The spatial degree of coherence affects the quality of imaging systems. Especially in microlithography and microscopy the image quality is optimized by choosing the "right" degree of coherence. Hence it is necessary to know how to adjust the desired degree of coherence. The illumination system of a microscope allows one to vary several parameters : field stop, aperture stop and Kohler/critical illumination. The purpose of this paper is to determine the influence of these parameters on the degree of coherence in the object plane of a usual illumination system. The degree ofcoherence is measured as variation of modulation of interference fringes, produced by a shear interferometer, with the shear distance. For any kind of illuminating aperture (circular, i.e. bright-field, annular, i. e. dark-field, rectangular and double slit) and for both Kohler and critical illumination we found a very good agreement with the concept of Hopkins' effective source, predicting a Fourier relationship between the intensity distribution in the pupil of the condenser and the degree of coherence in the object plane.

Confocal imaging is a coherent technique so that the amplitude and phase information can be extracted using interference techniques. A confocal interference microscope has been constructed based on a Michaelson interferometer. Phase changes of 25 mrad., corresponding to height changes of 1 nm. can be detected. The interferometer can be used to measure surface profiles, and also to measure aberrations in the optical system.

The addition of alternative, conventional non-confocal transmitted illumination to the tandem scanning reflected light microscope enables it to be used with all familiar and conventional modes of the microscope to discover a location of interest in a prepared sample, using the confocal modes when desired, or vice versa. Depth of field for the non-confocal transmitted light image can be controlled by the aperture of illumination. Conventional and confocal (coloured) images can be mixed in any proportion in the same frame.

The confocal scanning optical microscope (CSOM) has the major advantages over the standard microscope that it has extremely good range resolution and cross-sectioning capability, somewhat better transverse resolution, and is well adapted to quantitative measurements. The basic reason for the cross-sectioning capability is that the objective lens is illuminated from a collimated beam, as shown in Fig. la, which focuses the beam to a small spot on the object. The light reflected from the object then returns back through the objective lens and a beamsplitter, and is focused to illuminate a pinhole in front of the detector. The beam is defocused by moving the object out of the focal plane. Light at the pinhole is defocused and very little light gets back to the detector. Typically, with a large aperture lens, the 3 dB points of the response are of the order of 500 nm apart.

The confocal scanning optical microscope (CSOM) has the major advantages of a very short depth of focus, transverse definition, and image contrast that are better than with a standard microscope. The depth resolution of these microscopes is of the order of a wavelength. However, if it is necessary to carry out thickness measurements of films a small fraction of a wavelength thick, or profile steps with this order of height change, phase measurement techniques become a useful tool. Our aims have therefore been to incorporate within-CSOM phase contrast techniques and differential techniques for quantitative measurement of the position of edges, edge slope, and the thickness of thin films. In conventional microscopes this is most widely accomplished with Zernike phase contrast systems or by using Nomarski differential interference contrast (DIC). In CSOMs, heterodyne interference techniques have been used. This paper will describe two methods of phase imaging that we have been investigating, phase contrast and differential interference contrast. Most of the experiments were initially carried out on a single pinhole mechanically-scanned CSOM. Our recent goal has been to test our initial concepts on the real-time scanning optical microscope (RSOM). This microscope uses a Nipkow disk with 200,000 pinholes to dramatically increase the scan speed.

The tandem scanning reflected light microscope (TSRLM) is a confocal light microscope which has the capability of looking into living tissue and obtaining high resolution, high magnification images of cellular structure. TSRLM can be used to study living tissue such as all layers of the corneal epithelium including basal epithelial cells, keratocytes, nerves, inflammatory cells, bacteria, and corneal endothelium. For the first time in vision research, real-time, in vivo, microscopic images of normal and pathologic tissues can be obtained from human or animal eyes using the TSRLM. Compared to other methods of vital microscopy, TSRLM has no present rival. Specifically, TSRLM will: (1) Allow the hitopathologic analysis of living eyes, in vivo, over multiple observation periods without the need for tissue fixation and/or processing; (2) Assist in the acquisition and analysis of histopathologic images from human eyes, in vivo, in corneal disease; and (3) Greatly reduce the need for large numbers of animals in the histopathologic evaluation of experimental corneal disease and surgical procedures.

Many problems in ophthalmology deal with the three-dimensional structure of various parts of the living human eye. A complete confocal laser scanning microscope for eye diagnosis and first clinical results are described. Geometrical measurements of the cornea and topographical measurements of the retinal substructures are presented.

A prototype of a clinical optically sectioning, two-dimensional redox fluorescence imaging microscope is described. Ultraviolet light from an arc lamp is conducted to the optical system with a quartz optical fiber. A variable slit projects the light onto the cornea after passing an excitation interference filter. The dipping cone of the microscope applanates the cornea and focuses the light onto the endothelial cell layer which is 6 microns thick. The fluorescence emission from the mitochondrial reduced pyridine nucleotides (NADH + NADPH) is detected by a microchannel plate-gated intensifier attached to a Newvicon Video Camera and a digital image processor. The intrinsic natural cellular fluorescence is imaged and is indicative of the cellular state of oxidative metabolism. In addition, an optically sectioning microscope was developed with an optical spectrum analyzer to characterize the fluorescence spectra from thin layers in the cornea and ocular lens. Its unique feature is that the scanning objective is attached to a piezoelectric driver and scans the eye from the tear film to the aqueous humor. The depth resolution is 6 microns with an 100 x objective and 18 microns with a 50 power objective (100 micron slits). The applications include fluorescence measurements on biological layered structures. The present study involves the noninvasive measurement of oxidative metabolism of the component layers of the in vivo cornea. Finally, the utility of confocal microscopy in ophthalmology is demonstrated as a series of confocal images of the rabbit cornea with a depth resolution less than one micron.

A confocal beam scanning laser microscope (CBSLM) for confocal fluorescence and confocal reflection microscopy has been built at the European Molecular Biology Laboratory (EMBL) in Heidelberg. The instrument has now been used for almost one year with a large number of different biological specimens, experimental protocols and fluorophores. The instrument is stable, has a high detection efficiency and is easy to use.

A Type 2 (confocal) transmission laser scanning microscope containing dynamic mechanical testing facilities for the purpose of isolated muscle fiber studies is under development in our laboratory. An embryonic skeletal muscle cell may be kept alive and grown in a temperature-controlled chamber which is confined by two microscope objectives. The muscle cell can be electrically stimulated or manipulated using two motor clusters in 3 dimensions for imaging purposes while independent mechanical experiments are performed. The microscope consists of an interferometer which works either in a Mach-Zehnder configuration to provide cross-correlation functions from which magnitude, phase, and polarization information is obtained, or in a Michelson arrangement to provide auto-correlation functions (eventually for Fourier transform Raman spectroscopy). The microscope is presently controlled by a MicroVAX-II/GPX system while a dedicated parallel computer (multiple-instruction multiple-data) is being developed to cope with the demanding analysis and real-time control require-ments of the apparatus. The approach taken is to avoid acquiring 3-D images and instead to fit structural models to optical data acquired from the original object as needed by the model building algorithms.

Laser scanning microscopy was used to study dynamic processes in single cells. The laser scanning microscope of Heidelberg Instruments was complemented with a 1W krypton laser and a microinjection set-up. Simple algorithms were worked out which permit determination of local and integrated intensities in fluorescence scans. In one application the lateral diffusion of macromolecules was studied. The krypton laser was used to irreversibly photolyse fluorescently labeled dextrans in small volumes of a thin fluid layer; equilibration of the local fluorescence inhomogeneity by lateral diffusion was followed by repetitive scanning. In a second application the permeability of single red blood cell membranes which had been exposed to the complement cascade was studied. In a further application artificial nuclear proteins, constructed by molecular genetic methods, were injected into the cytoplasm of hepatoma cells. The kinetics of protein transport from cytoplasm to nucleus were derived from fluorescence scans. In all applications a good agreement between results obtained by laser scanning microscopy and those obtained independently by other methods and instruments was observed.

The investigation of epithelial cells with a confocal fluorescence microscope is only useful as long as the three dimensional structure of the sample is preserved. For many reasons it is not guaranteed that this goal can be achieved with fixed specimens. In order to perform relevant biological experiments one must find ways avoiding the "loss of information."

Aspects of the inverted versus top view configuration for confocal microscopy are examined with the inverted-type clearly offering advantages for high-resolution imaging of certain types of biological specimen. The role and possibilities of the use of pinholes of variable size in confocal microscopy is discussed and data are presented for combinations of objectives and various sizes of pinholes.

The new universal confocal LSM is a second-generation laser scanning microscope. This means, that laser scanning microscopy now made the transition from experimental set-up lab types to integrated workstations, where the manual handling of mechanical and optical components is left to the computer. The built-in microcomputer - now not only drives scanners and transforms signals into images but also controls directly the microscope functions. It turned out that this is a crucial step for making the LSM an universal instrument for widespread use in research and development. The switching from conventiona] microscopy to laser scanning modes and vice versa is performed by simply pressing keys. Not only images can be stored on the built-in hard disk but at the same time automati cally the corresponding set of parameters: Even weeks or months after creating an image the settings of the instrument belonging to this image can be called from the operators panel by loading a parameter file which defines the laser line used and its intensity setting, nosepiece position, zoom factor, averaging conditions, microscopy mode (transmitted, reflected or fluorescence) and parameters for signal conditioning. Since the microscope stand is motorized at a high degree, the computer recreates automatically the exact conditions desired after dialing the number of the parameter file. In this way working with the LSM becomes not only reproducible, but also the user is freed from the handling of mechanical parts and typing commands on a keyboard. Finally the automatized LSM allows true remote control by a host computer necessary for the most demanding 3D-reconstruction. The characteristics pointed out so far are prerequisites for the daily use by microscopists in life science, semiconductor research, development and testing and materials research.

The minority carrier distribution in a semi-infinite semiconductor due to light focussed onto the surface via a high numerical aperture lens is given. The contrast obtained in both Photoluminescence and Optical Beam Induced Current defect imaging is derived as a function of the illuminating lens numerical aperture. It is shown that the maximum resolution attainable in both techniques is comparable.

The minority carrier lifetime in gallium arsenide (GaAs) can be determined from time-resolved photolumi-nescence near the optical band edge, around 872 nm (1.42 eV) at room temperature. Spatial resolution of the order of a few micron is required for investigating microscopic fluctuations of he minority carrier lifetime in GaAs materials and in small, highly integrated devices. A photoluminescence lifetime spectrometer (PLS) based on the time-correlated single photon counting (TCSPC) technique was developed for this purpose. The instrument uses only solid state components, i.e. pulsed diode laser excitation and single photon avalanche diode (SPAD) detection, and is capable of measuring photoluminescence decay time constants of the order of 10 ps with 3 μm spatial resolution. Operation at a repetition rate above 50 MHz reduces the data collection time for a complete decay curve to a few seconds and thus permits one and two-dimensional scans to be done. The design of the PLS is discussed and first results from LEC grown GaAs substrates are presented.

We studied the lateral photo effect in CaAs/A1_xGa_1-xAs heterostructures both theoretically and experimentally. We observe a linear dependence of the photo voltage as a function of the position of the light spot. In our model this corresponds to a recombination length of the spatially separated electrons and holes longer than the length of the sample (1 mm). Deviations of this linear dependence are a direct indication of inhomogeneities in the conductive properties of the two-dimensional electron gas at the interface of the heterostructure. Results are shown in which long (1 mm) but very narrow cracks are seen.

The photoelectrochemical properties of semiconducting bismuth sulphide (Bi₂S₃) films (in contact with an aqueous electrolyte) grown on bismuth have been investigated. Using an Optically Beam Induced Contrast (OBIC) technique, it has been possible to image these films, and to identify local variations corresponding to grain boundaries and other recombination centres. It is seen that the form of these regions varies with the thickness of the film. Both object scanning and beam scanning have been investigated for generating images and these two methods are compared. Samples have also been investigated by Intensity Modulated Photocurrent Spectroscopy (IMPS). This method involves sinusoidal intensity modulation of the incident laser radiation at different frequencies, and the analysis of the resulting photocurrent response. Our extension of this method has been to map the IMPS response over the specimen surface in order to obtain specific information on local properties.

The laser probe of the scanning laser microscope generates electron-hole pairs in the IC semiconductor material which are separated at inversely-biased pn-junctions and can then be detected as photocurrent at external terminals. This OBIC (Optical Beam Induced Current) effect provides the physical basis for the localization and sensitivity measurements of the undesired latch-up in CMOS devices. The laser probe can also be used to measure logic-level time diagrams at the internal nodes of an IC. In addition, the scanning laser probe can be used to stimulate and localize malfunctions when the IC is operated at its marginal conditions.

A laser scanning microscope offers a non-destructive technique to locate and analyze latch-up in an IC. We have developed an advanced, fully automated latch-up analyzer coupled with the CAD system used for IC design. It consists of a laser scanning microscope, a x-y table for chip scanning, a monitor TV and a microprocessor based system for control of the test sequence and data analysis. Latch-up sensitivity is measured by stepwise increase of laser beam power using an acousto-optical modulator. The monitoring of the beam position and the modulator voltage while scanning the laser spot over the IC surface and the resulting current changes in the device's power supply locate the latch-up zone and its sensitivity. The sensitive regions found are overlaid graphically over the IC layout data to provide a redesign posibility. As an application example we consider a CMOS A/D converter IC and explain the system performance.

The infrared laser scan microscope is especially suitable for applications in material science and in the semiconductor industry. The infrared laser beam is deflected in x and y directions by a mirror system and the scanning beam is focussed with the help of a light microscope onto the sample. Four different IR (infrared) lasers can be used; the semiconductor lasers AlGaAs/GaAs with a wavelength of about 820nm and GaInAsP/InP with a wavelength of about 1.3μm and the helium-neon-lasers emitting at 1.152μm and 1.523pμm. The lasers are situated outside the optics and are connected by a special monomode glass fiber. The He-Ne laser, with a wavelength of 1.152μm, is of special significance in the investigation of silicon and ICs because its wavelength exactly corresponds to the energy gap of silicon at room temperature ( 1.07eV ). It is therefore possible to image silicon at different depths, to test devices from the back and, moreover, to produce an OBIC ( Optical Beam Induced Current ) signal from the back of the device.

A method is presented for calculating the reflectivity of a beam of arbitrary profile, in a prism coupler, which is generating plasmons on a non-uniform surface. The particular case of a line discontinuity is considered, and computations for specific, systems are presented. Implications for various methods of surface plasmon microscopy are discussed, including the use of image processing in a computerized system.

High resolution scanning reflection acoustic microscopy is a new scanning technique which provides information about the local elastic properties (both at the surface and in depth) of various kinds of objects. In the present work, two applications of the scanning system (Leitz ELSAM microscope, frequency range: 0.8-2.0 GHz) are considered. The first is the characterization of the adhesion of mouse neuroblastoma cells to a silicon substratum: rings of alternate intensities, originated by acoustic interference fringes, and shown in the cell image, are utilized to obtain information about cell morphology. In the second application, instead, acoustic microscopy is proposed as a non-destructive, inexpensive, and fast technique for characterizing semiconductor devices. The work is focused on the low-level processing (filtering, segmentation, and feature extraction) of the resulting acoustic images, to restore the original information and to measure several features useful in characterizing and understanding an object. The final goal is to determine the acoustic impedance and the acoustic attenuation of the object considered, and, in the case of living cells, to monitor them in time.

A novel semiconductor laser digital scanner composed of a monolithic semiconductor laser array and a conventional plastic lens is proposed. A high speed scanning, a simultaneous light emission, and a wide angle scanning are achieved. The semiconductor laser array consists of monolithically integrated ten index-guided 780nm emitters spaced by 0.3mm. The lens is an aspheric plastic lens which is widely used in the compact disc system with 4.5mm focal length and 0.45 N.A.. The maximum scanning angle is about 43 degrees and the beam diameter (FWHM) on a screen varies from 0.10mm to 0.53mm. Computer simulation using the ray tracing method is performed. We have found that the calculated results on the spheric lens case fit well with experimental ones using the aspheric lens.

The transmission near-field scanning technique can be used for determining the refractive index profile, the maximum core-cladding refractive index difference and/or numerical aperture, the geometrical characteristics and the mode-field dimensions in single-mode waveguides. In the experimental apparatus presented in the paper, the near-field pattern under test is magnified onto the faceplate of a vidicon camera, the video signal is sent to a computer--controlled video digitizer with single frame buffer memory organized as 512x512x8 bits. The incoming signal is digitized in real time to 8 bit resolution. Algorithms developed to opti-mize the scanning routine and to derive waveguide parameters from the measured data are described. As measured signals are usually noisy, techniques which reduce the noice level are also presented. Futhermore, techniques applied to compensate the vidicon sensitivity nonuniformity and nonlinearity are reported. Some examples of the results of measurements of the refractive index profiles and geometrical parameters like: core and cladding diameters, core and cladding non-circularities and core/cladding concentricity errors are presented.

The reflectance of Spanish manuscripts (paper and ink) dating from the XV to XIX centuries has been studied in the spectral range 350-1000 nm, specially when poor definition and/or manuscript degradation are present. The results have been applied to optimize the readability of the corresponding digital images of manuscript by choosing appropriately the spectral distribution of the source and the spectral sensitivity of the detector in a given digitizing device. For those manuscripts with severe degradation, the method gives enhanced original digital images that could be further processed resulting in optimal final images.

The paper contains a description of the principle of operation of a system to measure the position of marked points in a scene. The main application envisaged is in the study of human movement and biomechanics. This system eliminates the need for the time consuming computation involved in tracking particular points from scene to scene. Therefore many more points of interest may be studied than in existing video based systems. The implementation described uses a rotating disc that contains the scanning and sychronising pattern, an optical projection system and light sensors attached to the points of interest and connected to a desktop computer via an interface unit.