Large-image-format computed tomography imaging spectrometer for fluorescence microscopy.

Multispectral imaging has significantly enhanced the analysis of fixed specimens in pathology and cytogenetics. However, application of this technology to in vivo studies has been limited. This is due in part to the increased temporal resolution required to analyze changes in cellular function. Here we present a non-scanning instrument that simultaneously acquires full spectral information (460 nm to 740 nm) from every pixel within its 2-D field of view (200 ìm x 200 ìm) during a single integration time (typically, 2 seconds). The current spatial and spectral sampling intervals of the spectrometer are 0.985 ìm and 5 nm, respectively. These properties allow for the analysis of physiological responses within living biological specimens.

Fiber confocal reflectance microscope (FCRM) for in-vivo imaging.

In-vivo imaging can be achieved with a coherent-fiber-bundle based confocal reflectance microscope. Such a microscope could provide the means to detect pre-cancerous lesions in the cervix by characterizing cells' nuclear-to-cytoplasmic ratio. In this paper we present the design of such a fiber confocal reflectance microscope, with an emphasis on its optical sub-systems. The optical sub-systems consist of a commercially available microscope objective and custom designed telescope, scan lens, and coupling lens systems. The performance of the fiber confocal reflectance microscope was evaluated by imaging a resolution bar target and human cervical biopsy tissues. The results presented in this paper demonstrate a lateral resolution of 2 microm and axial resolution of 6 microm. The sensitivity of the system defined by the smallest refractive-index mismatch that can be detected is approximately Delta n 0.05.

UV light induced surface expansion phenomenon of hybrid glass thin films.

Liquid-phase deposition of sol-gel method derived hybrid glass materials is utilized for fabrication of UV-light-sensitive thin films. The hybrid glass material undergoes a surface-relief deformation when exposed to UV light. The observed deformation phenomenon is in the form of a physical expansion of the exposed areas. The UV light induced surface expansion of the hybrid glass film was used to fabricate near-sinusoidal diffraction gratings with periods of 24 microm, 18 microm, 12 microm, and 9 microm. The maximum deformation when the material was patterned as a diffraction grating was 0.685 microm. The hybrid glass material features an index of refraction of 1.52 at 632.8 microm, rms surface roughness of 2.2 +/- 0.8 microm after processing, and extinction coefficients of 1.2 x 10-3 microm-1 and 0.47 x 10-3 mm-1 at wavelengths of 633 nm and 1550 nm, respectively.

Telepathology overview: from concept to implementation.

Telepathology is the practice of pathology at a distance by using video imaging and telecommunications. Significant progress has been made in telepathology. To date, 12 classes of telepathology systems have been engineered. Rapid and ultrarapid virtual slide processors may further expand the range of telepathology applications. Next-generation digital imaging light microscopes, such as miniaturized microscope arrays (MMA), may make virtual slide processing a routine laboratory tool. Diagnostic accuracy of telepathology is comparable with that of conventional light microscopy for most diagnoses. Current telepathology applications include intraoperative frozen sections services, routine surgical pathology services, second opinions, and subspecialty consultations. Three telepathology practice models are discussed: the subspecialty practice (SSP) model; the case triage practice (CTP) model; and the virtual group practice (VGP) model. Human factors influence performance with telepathology. Experience with 500 telepathology cases from multiple organs significantly reduces the video viewing time per case (P < .01). Many technology innovations can be represented as S-curves. After long incubation periods, technology use and/or efficiency may accelerate. Telepathology appears to be following an S-curve for a technical innovation.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

Computed-tomography imaging spectrometer: experimental calibration and reconstruction results.

A temporally and spatially nonscanning imaging spectrometer is described in terms of computedtomography concepts, specifically the central-slice theorem. A sequence of three transmission sinusoidalphase gratings rotated in 60° increments achieves dispersion in multiple directions and into multiple orders. The dispersed images of the system's field stop are interpreted as two-dimensional projections of a three-dimensional (x, y, λ) object cube. Because of the size of the finite focal-plane array, this imaging spectrometer is an example of a limited-view-angle tomographic system. The imaging spectrometer's point spread function is measured experimentally as a function of wavelength and position in the field of view. Reconstruction of the object cube is then achieved through the maximum-likelihood, expectation-maximization algorithm under the assumption of a Poisson likelihood law. Experimental results indicate that the instrument performs well in the case of broadband and narrow-band emitters.

Ring-toric lens for focus-error sensing in optical data storage.

We discuss the design and performance of diffractive ring-toric lenses for focus-error sensing in optical data storage. A ring-toric lens images a point source of light to a ring-shaped image. Focus-error sensing is accomplished by means of monitoring the change in ring radius: The ring expands in response to a diverging wave front, and the ring contracts in response to a converging wave front. We describe the use of a segmented phi detector to generate a focus-error signal (FES). We found that the FES slope, a measure of sensitivity to disk defocus, is higher for the ring-toric lenses described in this paper than for other techniques such as the astigmatic and the obscuration methods. We measured an FES slope of 0.7 per micrometer of disk defocus (microm(-1)). The corresponding theoretical FES slope is 0.96 microm(-1).

Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor.

We present a technique to measure the wavefront in the exit pupil of a microscope to determine the microscope's three-dimensional point spread function (PSF) experimentally. The wavefront yields the microscope PSF through a Fourier transform that models propagation of light from the exit pupil to the image plane. A Shack-Hartmann wavefront sensor is used to measure the wavefront shape by recording lateral displacements of a grid of focused spots created by a lenslet array. The displacement of each spot is related to the local wavefront slope. Thus, with appropriate sampling across the exit pupil, the entire wavefront can be reconstructed. This technique does not require the use of a sub-resolution object to obtain the three-dimensional microscope PSF. Consequently, larger, brighter fluorescent objects may be imaged, thereby reducing the requirements for detector sensitivity and leading to a three-fold increase in the axial range over which the PSF is measured. The Shack-Hartmann technique results in a description of the PSF as a continuous function whose sampling is not dependent on the size of the CCD pixels. The Shack-Hartmann sensor is not limited by the numerical aperture of the objective and can easily be calibrated to measure the PSF at any wavelength.

Binary-phase zone-plate arrays based on hybrid solgel glass.

An organically modified silane zirconate-based solgel material is used for the fabrication of binary-phase zone-plate arrays. The synthesized hybrid solgel material has a negative tone under UV exposure and can be patterned by a UV-lithography process. The transmittance of the material is nearly 100%, and the refractive index is 1.52. Two different diffractive lens arrays with focal lengths of 5 and 42 cm have been fabricated. The average roughness of the zone surface is less than 20 nm. The diffraction efficiencies of the lens arrays are measured as a function of modulation depth and exposure dose. A diffraction efficiency of 30% is achieved.

Multilayer-coating-induced aberrations in extreme-ultraviolet lithography optics.

A multilayer coating alters the amplitude and phase of a reflected wave front. The amplitude effects are multiplicative and well understood. We present a mathematical formalism that can be used to describe the phase effects of coating in a general case. On the basis of this formalism we have developed an analytical method of estimating the wave-front aberrations introduced by the multilayer coating. For the case of field-independent aberrations, we studied both uniform and graded multilayer coatings. For the case of field-dependent aberrations, we studied only the effects of a uniform multilayer coating. Our analysis is based on a coated plane mirror tilted with respect to an incident converging beam. Altogether we have found, up to the second order, the following aberrations: a field-dependent piston, a field-squared-dependent piston, defocus, field-independent tilt, field-independent astigmatism, and anamorphic magnification. To obtain numerical results we apply our analysis to the specific case of a plane mirror tilted 8.2 deg with respect to an incident converging beam with a numerical aperture of 0.1. We find that the magnitudes of the field-independent aberration coefficients for the graded coating are approximately ten times smaller than those for the uniform coating. We show that a coating can introduce anamorphic magnification.

Computed tomography-based spectral imaging for fluorescence microscopy.

The computed tomography imaging spectrometer (CTIS) is a non-scanning instrument capable of simultaneously acquiring full spectral information (450-750 nm) from every position element within its field of view (75 microm x 75 microm). The current spatial and spectral sampling intervals of the spectrometer are 1.0 microm and 10 nm, respectively. This level of resolution is adequate to resolve signal responses from multiple fluorescence probes located within individual cells or different locations within the same cell. Spectral imaging results are presented from the CTIS combined with a commercial inverted fluorescence microscope. Results demonstrate the capability of the CTIS to monitor the spatiotemporal evolution of pH in rat insulinoma cells loaded with SNARF-1. The ability to analyze full spectral information for two-dimensional (x, y) images allows precise evaluation of heterogeneous physiological responses within cell populations. Due to low signal levels, integration times up to 2 s were required. However, reasonable modifications to the instrument design will provide higher system transmission efficiency with increased temporal and spatial resolution. Specifically, a custom optical design including the use of a larger format detector array is under development for a second-generation system.

Optimization of retardance for a complete Stokes polarimeter.

We present two figures of merit based on singular value decomposition, which can be used to assess the noise immunity of a complete Stokes polarimeter. These are used to optimize a polarimeter featuring a rotatable retarder and a fixed polarizer. A retardance of 132 degrees (approximately three-eighths wave) and retarder orientation angles of +/-51.7 degrees and +/-15.1 degrees are found to be optimal when four measurements are used. Use of this retardance affords a factor-of-1.5 improvement in signal-to-noise ratio over systems employing a quarter-wave plate. A geometric means of visualizing the optimization process is discussed, and the advantages of the use of additional measurements are investigated. No advantage of using retarder orientation angles spaced uniformly through 360 degrees is found over repeated measurements made at the four retarder orientation angles.

Midwave-infrared snapshot imaging spectrometer.

We report results from a demonstration of a midwave-infrared, nonscanning, high-speed imaging spectrometer capable of simultaneously recording spatial and spectral data from a rapidly varying target scene. We demonstrated high-speed spectral imaging by collecting spectral and spatial snapshots of blackbody targets and combustion products. The instrument is based on computed tomography concepts and operates in a midwave-infrared band of 3.0-5.0 mum. We record raw images at a frame rate of 60 frames/s, using a 512 x 512 InSb focal-plane array. Reconstructed object cube estimates were sampled at 46 x 46 x 21 (x, y, lambda) elements, or 0.1-mum spectral sampling. Reconstructions of several objects are presented.

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved.

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 micro m and axial resolution is 6 micro m. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s-1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.

Microtags with 150-nm line gratings fabricated by use of extreme-ultraviolet lithography.

The microtag concept is an anticounterfeiting and security measure. Microtags are computer-generated holograms (CGH's) consisting of 150-nm lines arranged to form 300-nm-period gratings. The microtags that we describe were designed for readout at 442nm . The smallest microtag measures 56micromx80 microm when viewed at normal incidence. The CGH design process uses a modified iterative Fourier-transform algorithm to create either phase-only or phase-and-amplitude microtags. We also report on a simple and compact readout system for recording the diffraction pattern formed by a microtag. The measured diffraction patterns agree very well with predictions.

High-speed spectral imager for imaging transient fluorescence phenomena.

We describe fluorescence spectral imaging results with the microscope computed-tomography imaging spectrometer (muCTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, muCTIS can be used to image dynamic phenomena. The results presented consist of proof-of-concept imaging results with static targets composed of 6-mum fluorescing microspheres. Image data were collected with integration times of 16 ms, comparable with video-frame-rate integration times. Conversion of raw data acquired by the muCTIS to spatial and spectral data requires postprocessing. The emission spectra were sampled at 10-nm intervals between 420 and 710 nm. The smallest spatial sampling interval presented is 1.7 mum.

Demonstration of a high-speed nonscanning imaging spectrometer.

We report results from a field demonstration of a nonscanning high-speed imaging spectrometer [computed-tomography imaging spectrometer (CTIS)] capable of simultaneously recording spatial and spectral information about a rapidly changing scene. High-speed spectral imaging was demonstrated by collection of spectral and spatial snapshots of a missile in flight. This instrument is based on computed-tomography concepts and operates in the visible spectrum (430-710nm). Raw image data were recorded at video frame rate (30frames / s) and an integration time of 2ms. An iterative reconstruction of the spatial and spectral scene information from each raw image took 10s. We present representative missile spectral signatures from the missile firing. The accuracy of the high-speed spectrometer is demonstrated by comparison of extended-source static-scene spectra acquired by a nonimaging reference spectrometer with spectra acquired by use of CTIS imaging of the same static scenes.

Demonstration of a computed-tomography imaging spectrometer using a computer-generated hologram disperser.

We have constructed a computed-tomography imaging spectrometer that uses a phase-only computer-generated hologram (CGH) array illuminator as the disperser. This imaging spectrometer collects multiplexed spatial and spectral data simultaneously and can be used for flash spectral imaging. The CGH disperser has been designed to maintain nearly equal spectral diffraction efficiency among a 5 x 5 array of diffraction orders and to minimize diffraction efficiency into higher orders. Reconstruction of the (x, y, lambda) image cube from the raw, two-dimensional data is achieved by computed-tomography techniques. The reconstructed image and spectral-signature data compare favorably with measurements by other spectrometric methods.

Mass-producible microscopic computer-generated holograms: microtags.

We have developed a method for encoding phase and amplitude in microscopic computer-generated holograms (microtags) for security applications. An 8 x 8 cell phase-only and an 8 x 8 cell phase-and-amplitude microtag design has been exposed in photoresist by the extreme-ultraviolet (13.4-nm) lithography tool developed at Sandia National Laboratories. Each microtag measures 80 microm x 160 microm and contains features that are 0.2 microm wide. Fraunhofer zone diffraction patterns can be obtained from fabricated microtags without any intervening optics and compare favorably with predicted diffraction patterns.