Optical laser-feedback microscopy (LFM), a scanning confocal interference microscopy, furnishes nanometer axial and 200 nm lateral resolution of surface topology when examining well-defined reflective hard surfaces such as semiconductors, metals, or other materials. Biological samples (e.g., cells or tissues under physiological conditions) are important objects for examination by LFM as the improved resolution available with this new method of optical microscopy can furnish biological structural information on a scale previously only attainable with electron microscopy but without the necessity for sample fixation or staining or the effect of ionization-produced radiation damage. Although the small refractive-index changes that occur at biological-membrane/water interfaces produce sufficient signal to be useful in LFM- imaging, being able to obtain `optical-sectioning' at a scale of nanometers and derive 3D information at that resolution would allow intracellular biological structures (e.g., organelles, chromosomes) and their functional changes to be determined on physiological-viable samples. By incorporating information from two simultaneously-acquired LFM images (optical phase and amplitude reflectivity), information has been obtained on a variety of biological cells; two examples will be presented, images of a green algae (Chlamydomonas reinhardtii) and of a human erythrocyte.

A new confocal scanning beam laser microscope/macroscope is described that combines the rapid scan of a scanning beam laser microscope with the large specimen capability of a scanning stage microscope. This instrument combines an infinity-corrected confocal scanning laser microscope with a scanning laser macroscope that uses a telecentric f^*(theta) laser scan lens to produce a confocal imaging system with a resolution of 0.25 microns at a field of view of 50 microns to 5 microns at a field of view of 75,000 microns. The frame rate is 3 seconds per frame for a 512 X 512 pixel image, and 45 seconds for a 2048 X 2048 pixel image. Changes made in the instrument to increase the image capture from 512 X 512 pixels to 2048 X 2048 pixels are described. Applications discussed focus on three important advantages of the instrument over a confocal scanning laser microscope: an extremely wide range of magnification, the ability to record very large data sets, and the ability to image very large specimens. Examples are presented from imaging of fibers in paper, latent fingerprint detection, and reflected-light and photoluminescence imaging of porous silicon.

We first review a newly developed 3D imaging technique called optical scanning holography (OSH), and discuss recording and reconstruction of a point object using the principle of OSH. We then derive 3D holographic magnification, using three points configured as a 3D object. Finally, we demonstrated 3D imaging capability of OSH by holographically recording two planar objects at different depths and reconstructing the hologram digitally.

We present an algorithm for estimating nonrigid motion of chromosomes in 4D microscopic images. Chromosomes are represented by a graph and motion estimation is formulated as a graph matching problem. All chromosomes within the graph are located, and then simulated annealing is used to find the mapping of chromosomes at time t onto chromosomes at time t+1 that minimizes the integrated displacement along each chromosome. Results with actual 4D images indicate that this model-based approach is sufficiently robust to correctly track the motion of chromosomes under conditions of limited spatial and temporal resolution. Using the motion estimate, previously developed methods for the quantitative analysis of 3D structure are extended to four dimensions, allowing chromosome mobility, flexibility, and interactions to be measured. Application of these algorithms to 4D images of Drosophila metaphase chromosomes in vivo allows visualization of clearly defined domains of high chromosomal flexibility, as well as other regions of distinctly lower chromosomal mobility which may coincide with centrometers.

Fluorescence in situ hybridization (FISH) is useful for analyzing specific nucleic acid sequences in individual cells. Its application to tissue sections has been limited however because of the difficulties of performing the hybridization and analysis in sections that are thick enough to contain intact nuclei. Recent improvements in FISH permit hybridization with chromosome-specific, centromeric probes throughout 20 micrometers formalin fixed, paraffin- embedded sections, which do contain many intact nuclei. This paper describes software to facilitate analysis of these 3D hybridizations. We have developed two algorithms for analyzing 3D, confocal images of thick sections. One displays 2D, maximum-intensity, projection images through the original 3D image at different angles. When projections are viewed sequentially, the 3D image appears semi-transparent and rotates. The second algorithm allows interactive enumeration of FISH signals. Each signal is marked by the analyst. Then, for each pair of marked signals, a 2D slice image along the line connecting both marked signals and parallel to the z (depth) axis is displayed. From this slice, the analyst decides if the signals are in the same or different nuclei, or if the signals should be rejected because they are in a nucleus truncated by the upper or lower surface of the section. After consideration of all pairs of signals, the algorithm produces a map of the tissue section showing the numbers of signals in each of the intact nucleus. The algorithms enable analysis of small, premalignant and early malignant lesions and infiltrative lesions that cannot be analyzed by other molecular techniques and permit the direct correlation of FISH information with histology/cytology.

A method to record the capillary networks in glomeruli and lungs has been developed. This method includes a new technique for recording the specimen and new algorithms for analysis of the recorded volumes. When recording a dense specimen, the usefully attainable depth is often limited by absorption and scattering by material above the plane of focus. We found this to be a problem when studying lung and kidney tissue. A solution based on successive recording and removal of material from the specimen was developed which enabled the recording of thick layers (several hundred microns). Using a custom-made milling device, we recorded datasets of both lungs and kidneys. We then traced the capillary networks using a seed algorithm that reduces the risk of bleeding out into space not belonging to the region being filled. With this method the understanding of the specimen structure is greatly enhanced, and quantitative data can be collected from the capillary networks.

A method for confocal optical microscopy is presented in which the role usually played by spatial modulators (pinhole or fiber) placed before the detector, is electronically emulated by an adaptive pixel-exclusion process performed at the level of an image detector. The technique is optimally suited to the construction of systems based on a multi-point illumination and detection approach, with the aid of conventional lamps and CCD image sensors. Due to its inherent simplicity and versatility, the proposed design can be efficiently employed in transmission, reflection and fluorescence microscopy, using trans- or epi-illumination configurations. The resulting advantages appear particularly relevant in biomedical applications, not only with the purpose of setting up practical and affordable instruments, but also in order to avoid specimen damage or signal saturation effects, as well as to obtain confocal images of light-absorbing microscopic structures.

This paper presents a method of automatic segmentation in sequence medical tissue slice images with high noise. A seed growing method which is constrained by the parameter table is used to detect edge. Priority and step parameter are used to adaptive adjust the detection. The paper described the mechanism to change the priority and detect parameter and their step in detail. The authors designed an evaluation function to search the state space.

We discuss the differences between polarization contrast imaging in conventional and confocal systems and show that because of the fundamentally different imaging properties of the two systems, the extinction coefficient is non-zero in a conventional system even with perfect polars. We then discuss the axial response of high numerical aperture systems and show that a polarization effect leads to an asymmetric response when the objective lens numerical aperture is greater than the refractive index of the specimen.

Some time ago we described a new type of near-field imaging system using the solid immersion lens. In this paper we shall discuss how this type of lens can be employed in optical storage systems to increase the bit density, along with the research that has been carried out at Stanford in cooperation with Sony Corporation and IBM, Almaden to reach this goal. 12 ()j aimis to work with both CD ROMs and magneto-optical storage.

Recently, several techniques have been proposed for sectioning deep surface relief using the narrow coherence function of white light interference fringes, classified under the general heading of `coherence microscopy'. The maximum value of the coherence function is used as a probe plane at a known height of Z(x,y), which is scanned over the depth of the sample. Nanometric vertical resolution over a range of many tens of microns is attainable by this means. An interesting feature of coherence microscopy is that the high depth discrimination due to the temporal coherence of the illumination beam also leads to super-resolution laterally. This feature is useful in many microelectronics applications where there is a requirement for profiling structures such as grooves, wires and technological layers at a submicron scale laterally and with a nanometric resolution vertically over a depth of many microns. In this work, we present results using coherence microscopy for profiling a variety of micron sized structures with near 0.2 micrometers lateral resolution over a depth of field of 5 micrometers . Comparisons are made with SEM and AFM in order to raise some of the issues involved in a correctly interpreting high resolution synthetic images.

We measure the form of the point spread function in the three main configurations of 4 Pi microscopy as well as the traditional confocal arrangement. We confirm, for the first time experimentally, the equivalence between the 4 Pi (A) and 4 Pi (B) geometries. We measure a 6.9 fold increase in axial resolution over the confocal case.

When a confocal fluorescence microscope with a high numerical aperture oil immersion objective is focused deep into an aqueous medium, aberrations result which weaken and blur the observed image. We have designed, fabricated and tested a two-level binary phase mask which partially corrects these aberrations, improving image brightness and resolution. A four- level mask was also designed, fabricated and tested with further brightness and resolution improvement. The mask designs and simulated and measured results are presented in this paper.

Fluorescent molecules having single-photon absorption in the blue and the UV can be excited with infra-red light via a process known as two-photon excitation. The combination of this technique with scanning techniques can be exploited for 3D microscopic imaging. The two- photon process is confined to a restricted volume in the sample determined by the laser focus, resulting in inherent confocality. Other advantages are reduced photo-bleaching of the samples and a larger penetration depth of the excitation light. The implementation of time-gated detection techniques allows fluorescent lifetime imaging. This drastically improves the selectivity and contrast of the images.

A new technique for the measurement of fluorescence lifetimes relies on the (near steady state) excitation with short optical pulses. The novel technique has the potentiality to provide high time resolution--since it relies on the laser pulse duration, rather than on electronic gating techniques--and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. Combined with confocal microscopy it enables the spatial determination of the fluorescence lifetimes, the value of which is influenced by the local environment of fluorescent probe molecules in biological samples. The principle of the technique is discussed within a theoretical framework taking into account various secondary effects.

We here report on using fluorescence life times recordings in confocal microscopy to detect individual fluorophores in multiple stained tissue. Using indirect fluorescence immunohistochemistry with secondary antisera conjugated to the fluorophores Lissamine Rhodamine (LRSC), Texas Red or Cyanine 3.18 (Cy-3); one, two or three epitopes were labelled in tissues from rat spinal cord and dorsal root ganglia. The tissue sections were examined and analyzed in a beam-scanning confocal microscope equipped with devices for fluorescence lifetime measurements. The results show that fluorophore life-times can be used to separate two or more fluorophores in individual axon terminals, provided that the fluorophore labelling is strong enough and the life-times of the deployed compounds are sufficiently separate. Thus, the presence of Cy-3, LRSC and Texas Red, as well as mixtures of these compounds could be detected in individual tissue profiles. Contribution by tissue autofluorescence was unmistakably identified by its complex multiexponential emission decay. We also show that fluorescence lifetimes differs between antiserum-conjugates for one and the same fluorophore, and the possibility to use fluorophore life-times to probe chemical environmental changes in situ is discussed. The implementation of a life-time recording device in a confocal microscope has the advantage of providing a good spatial resolution. Furthermore, by deploying fluorophores emitting within the same wavelength band, problems due to chromatic errors will be completely avoided.

A spectrometer has been developed for the Phoibos confocal scanning laser microscope. With this spectrometer, spectral information from a single point, or a user defined region, within the microscope specimen, can be recorded. The spectrometer is based on an integrated spectrometer module, manufactured by Carl Zeiss, Germany. The module takes its light input signal through a fiber with an entrance diameter of 0.5 mm. Integrated in the spectrometer module are dispersing optics, based on a grating, as well as preamplifier electronics. A regulated cooling unit keeps the detector at -4 degree(s)C, thereby allowing longer integration times. The spectral resolution, defined as the minimum distance between two peaks (Rayleigh criterion) is approximately 10 nm. The entrance of the optical fiber is employed as a pinhole. With different magnification in the optical path leading the light to the spectrometer, the entrance can either be employed as a pinhole of the same size as the one used during conventional confocal scanning, i.e. the 3D spatial resolution will be retained, or the light throughput can be increased at the expense of optical resolution. With the described equipment, studies of rodent lung and liver specimens containing porphyrins have been made. Organs from animals injected with (delta) -amino levulinic acid, a precursor to protoporphyrin IX and haem in the haem cycle, have been studied. Spectroscopic detection is necessary in order to separate the porphyrin signal from other fluorescent components in the specimen.

In 3D fluorescence microscopy, a series of 2D images is collected at different focal settings through the specimen. Each image in this series contains the in-focus plane plus contributions from out-of-focus structures that blur the image. Furthermore, as the series is collected the fluorescent dye in the specimen fades over time in response to the total excitation light dosage which progressively increase as more optical slices are collected. Thus the different optical slices are 2D images of different 3D objects, in the sense that at each time point, the object has a different overall intensity. To date, the approach to compensate for this decay has been to precondition the image by dividing the intensities in each optical slice by a decaying exponential before processing the image by any of a number of existing deblurring algorithms. We have now directly incorporated fluorescent decay into maximum-likelihood estimators for the 3D distribution of fluorescent dye. We derived a generalized expectation-maximization algorithm for the simultaneous estimation of the decay constant, considered homogeneous, and the distribution of fluorescent dye.

A weakness of standard 3D microscopies--both confocal and widefield+deconvolution-- is that their resolution is substantially worse in the axial direction than in the lateral plane. We describe two new widefield techniques with substantially improved axial resolution that actually exceeds the lateral resolution. As is well known, the resolution is related to the angle over which the objective lens collects light. In our first technique, light is collected over an enlarged set of angles by using two objective lenses on opposite sides of the sample. The two image beams are combined coherently on the same CCD camera. Interference between the beams yields new, previously inaccessible sample information. The second technique applies a similar concept to the illumination light in fluorescence microscopy. Light from an extended, spatially incoherent light source--such as a standard arc lamp--is split and directed through the two opposing objective lenses so as to create a narrow interference fringe at the focal plane in the sample. This spatial structure in the excitation light yields access to new sample information. The two techniques can easily be used together; the combined technique promises an axial resolution improvement of a factor of seven over standard widefield microscopy.

Two-photon excited fluorescence microscopy was used to study unstained tissue and paper samples. As an excitation source a mode-locked Ti:Sapphire laser was utilized. In the experiments we used a conventional fluorescence microscope with a scanning board. The incoming laser pulses were focused onto the sample and the epifluorescence observed. In the spectroscopic measurements the fluorescence light was projected either on the slit of an polychromator with a CCD camera or, in some experiments, on a streak camera connected to the polychromator. The signal was then detected by a 2D-CCD camera. Fluorescence images were scanned by recording the fluorescence light pixel by pixel with a photomultiplier tube. Signal filtering and image processing were performed on a personal computer. Tissue samples from animals treated with photodynamic therapy were examined. The tissue contained protoporphyrin IX as a photosensitizer.

The nature of 3D imaging with confocal optical system using principles of tomography and geometrical optics is analyzed. Image intensity in any plane of image space is described by the 2D section of a 3D summary image of the sample is obtained.

Because of the quickly growing demand to handle micro- and nanostructures, micro- and nanorobotics has become an active field of research in the last years. One major problem of this new technology is how to build a sensor system that can control the robot motion within the required accuracy. For the micro and nano domain we specified an accuracy of one micro and ten nanometers respectively. We employ light-optical and Scanning Electron microscopes as they have the advantage that the robot and the target objects can be observed and positioned within a larger field of view. This renders nearly real-time processing possible. The strong requirements for accuracy and reliability demand a thorough sensor calibration. We propose a versatile photogrammetric approach for 3D calibration of both microscope types. We begin with a critical review of existing macroscopic and microscopic sensor models. Then we establish an algorithm using multi stereo geometry. This framework not only provides a highly accurate estimate of the mapping function from work space to image space, but also compensates image distortions caused by the lens system and the frame-grabber. The mapping function is further analyzed with respect to accuracy and determinability and non-significant parameters are eliminated. Numerical results based on simulated data are discussed.

Point projection microradiography has established value for imaging large, wet, opaque, and intact specimens in 2D projection views. We have developed a 3D microtomography system by combining the principles of microradiography with computed tomography (CT). An extension of conventional CT methods is utilized to yield 3D data from 2D microradiographic projections. Use of 2D cone beam projections rather than 1D projections of a slice simplifies the specimen motion hardware, and reduces the amount of wasted radiation. Our imaging system consists of a microfocus x-ray source and x-ray image intensifier coupled to a CCD camera. The system is flexible in the size of specimens which can be imaged. Resolving power varies with specimen size from 4 lp/mm for 50 mm diameter objects to 40 lp/mm for 3 mm diameter objects. Image resolution is isotropic in three dimensions. The 3D nature of the resulting image data can be used to visualize internal structure and compute stereologic parameters such as volume, surface area, and surface/volume orientation. This instrument has been used to image bone specimens in studies of human vertebrae, human femoral necks, dog metacarpals, and rabbit tibias. Other applications include imaging small industrial parts, plastics, ceramics, composite materials, and geologic specimens.

Biological soft x-ray microscopy using x-ray optics and synchrotron sources has made possible quantitative, element-specific imaging of whole cells in aqueous media at significantly higher resolutions than those of conventional visible light methods. Tomographic reconstruction has been proposed as a means to realize the full potential of the method for viewing thick objects whose structures would otherwise be superimposed in single view projections. The authors present an iterative tomographic reconstruction algorithm, using a regularized weighted least squares objective function, accelerated with the conjugate gradient approach, and modified for the problem of transmission tomography with correction of blurring by an instrumental point spread function. The non-negativity constraint is implemented using a preconditioner. We show by computer simulations that reconstructions that meet realistic and acceptable goals for spatial and density resolution should be achievable at doses compatible with the structural integrity of biological samples at the specific resolution.

Proper characterization of surface topography is critical for understanding a wide range of chemical and biological processes at the interface. This paper describes a method for characterizing the complexity of a surface from its depth map acquired with a laser vision sensor. The technique used to calculate fractal dimension was based on the variation method. Instead of trying to estimate the dimension around 0 however, this was done at specific scales over a range of values. We will show the impact of using local information to estimate the complexity of the surface and emphasize the problems that can be encountered when blindly trying to estimate the fractal dimension of 3D data. We conclude that linear local approximation of the object should be used to quantify its complexity, and to determine the scale at which analysis should be done. The method was applied to two samples of plasma coated titanium plates generated under differenty spraying conditions. The results show that our technique can provide a quantification scheme for standardization of the coating process and can improve the validation of manufacturing technologies.

Confocal microscopy systems can be linked to 3D data oriented devices for the interactive navigation of the operator through a 3D object space. Sometimes, such environments are named `virtual reality' or `augmented reality' systems. We consider optical confocal laser scanning microscopy images, in fluorescence with various excitations and emissions, and versus time The aim of our study has been the quantitative spatial analysis of confocal data using the false-color composition technique. Starting from three 2D confocal fluorescent images at the same slice location in a given biological specimen, a new single image representation of all three parameters has been generated by the false-color technique on a HP 9000/735 workstation, connected to the confocal microscope. The color composite result of the mapping of the three parameters is displayed using a resolution of 24 bits per pixel. The operator may independently vary the mix of each of the three components in the false-color composite via three (R, G, B) mixing sliders. Furthermore, by using the pixel data in the three fluorescent component images, a 3D space containing the density distribution of these three parameters has been constructed. The histogram has been displayed in stereo: it can be used for clustering purposes from the operator, through an original thresholding algorithm.

We have developed digital 3D Fourier transform methods for comparing the 3D spatial frequency content and hence the axial and transverse resolution of confocal versus conventional microscope images. In particular, we have utilized these techniques to evaluate the performance of our recently-developed confocal transmission microscope for bright field and Nomarski DIC imaging. We have also found that Fourier methods, such as the Hibert transform, can be successfully employed to overcome the difficulty of visualizing differentially-shaded phase objects, in 3D, that have been acquired using transmission DIC optics.

`Volume imaging' is the process of visualizing image data that exists on a grid in three- dimensions: at every point in the volume, a gray-level or color value is known. These image volumes usually result from building a `stack' from a sequence of cross-sectional views, such as those from CT, MRI, and confocal imaging. Stereoscopic viewing is a very effective way of viewing and analyzing these volumes. The stereo pair is constructed using ray projections. My objectives for this paper are three-fold: (1) To present a new way to view stereo images on a PC computer. (2) To demonstrate the ease-of-use and high image quality of the system using sample images from confocal microscopy. (3) To present preliminary results for projection image processing on a PC from CT and MRI image stacks.