Binary pseudo-random patterned structures for modulation transfer function calibration and resolution characterization of a full-field transmission soft x-ray microscope.

We present a modulation transfer function (MTF) calibration method based on binary pseudo-random (BPR) one-dimensional sequences and two-dimensional arrays as an effective method for spectral characterization in the spatial frequency domain of a broad variety of metrology instrumentation, including interferometric microscopes, scatterometers, phase shifting Fizeau interferometers, scanning and transmission electron microscopes, and at this time, x-ray microscopes. The inherent power spectral density of BPR gratings and arrays, which has a deterministic white-noise-like character, allows a direct determination of the MTF with a uniform sensitivity over the entire spatial frequency range and field of view of an instrument. We demonstrate the MTF calibration and resolution characterization over the full field of a transmission soft x-ray microscope using a BPR multilayer (ML) test sample with 2.8 nm fundamental layer thickness. We show that beyond providing a direct measurement of the microscope's MTF, tests with the BPRML sample can be used to fine tune the instrument's focal distance. Our results confirm the universality of the method that makes it applicable to a large variety of metrology instrumentation with spatial wavelength bandwidths from a few nanometers to hundreds of millimeters.

Synchrotron-Based FTIR Spectromicroscopy: Cytotoxicity and Heating Considerations.

Synchrotron radiation-based Fouriertransform infrared (SR-FTIR)spectromicroscopy is a newly emergingbioanalytical and imaging tool. This uniquetechnique provides mid-infrared (IR)spectra, hence chemical information, withhigh signal-to-noise at spatial resolutionsas fine as 3 to 10 microns. Thus it enablesresearchers to locate, identify, and trackspecific chemical events within anindividual living mammalian cell. Mid-IRphotons are too low in energy (0.05-0.5eV) to either break bonds or to causeionization. In this review, we show thatthe synchrotron IR beam has no detectableeffects on the short- and long-termviability, reproductive integrity,cell-cycle progression, and mitochondrialmetabolism in living human cells, andproduces only minimal sample heating (<0.5°C). These studies haveestablished an important foundation forSR-FTIR spectromicroscopy in biological andbiomedical research.

Very High Power THz Radiation Sources.

We report the production of high power (20watts average, ∼ 1 Megawatt peak) broadbandTHz light based on coherent emission fromrelativistic electrons. Such sources areideal for imaging, for high power damagestudies and for studies of non-linearphenomena in this spectral range. Wedescribe the source, presenting theoreticalcalculations and their experimentalverification. For clarity we compare thissource with one based on ultrafast lasertechniques.

Observation of broadband self-amplified spontaneous coherent terahertz synchrotron radiation in a storage ring.

Bursts of coherent synchrotron radiation at far-infrared and millimeter wavelengths have been observed at several storage rings. A microbunching instability has been proposed as the source for the bursts. However, the microbunching mechanism has yet to be elucidated. We provide the first evidence that the bursts are due to a microbunching instability driven by the emission of synchrotron radiation in the bunch. Observations made at the Advanced Light Source are consistent with the values predicted by the proposed microbunching model. These results demonstrate a new instability regime for high energy synchrotron radiation sources and could impact the design of future sources.

IR spectroscopic characteristics of cell cycle and cell death probed by synchrotron radiation based Fourier transform IR spectromicroscopy.

Synchrotron radiation based Fourier transform IR (SR-FTIR) spectromicroscopy allows the study of individual living cells with a high signal to noise ratio. Here we report the use of the SR-FTIR technique to investigate changes in IR spectral features from individual human lung fibroblast (IMR-90) cells in vitro at different points in their cell cycle. Clear changes are observed in the spectral regions corresponding to proteins, DNA, and RNA as a cell changes from the G(1)-phase to the S-phase and finally into mitosis. These spectral changes include markers for the changing secondary structure of proteins in the cell, as well as variations in DNA/RNA content and packing as the cell cycle progresses. We also observe spectral features that indicate that occasional cells are undergoing various steps in the process of cell death. The dying or dead cell has a shift in the protein amide I and II bands corresponding to changing protein morphologies, and a significant increase in the intensity of an ester carbonyl C===O peak at 1743 cm(-1) is observed. Biopolymers (Biospectroscopy) 57: 329-335, 2000

Equivalence of focusing conditions for holographic and varied line-space grating systems.

The focal conditions for diffraction grating systems that employ holographic and varied line-space gratings are shown to be formally identical. An example is given in which the recording geometry of a holographic grating is derived that provides the same imaging (to lowest order) as a ruled grating whose grooves, while straight and parallel, vary in spacing across the surface of the grating.

Numerical design method for aberration-reduced concave grating spectrometers.

A general method is described for the design of single concave grating optical systems. Proper parameter and variable sets are defined, and a straightforward technique leads to a final set of optimized variable values which minimize a merit function based on spectroscopic performance.

Excitation under the scanning electron microscope of DNA-associated fluorescence from chicken erythrocyte nuclei, polytene chromosomes and adenovirus 2 virions.

Biological structures not seen by conventional light microscopy, such as longitudinal striations in polytene chromosomes, and, at the limit of sensitivity, virions of adenovirus 2, have been detected via DNA-associated fluorescence excited under the scanning electron microscope. The maximum sensitivity realized, about 1 detected photon per 700 base pairs, falls short by about an order of magnitude of that required to achieve, in unreplicated specimens, the 2 nm intrinsic resolution of the method. A combination of D2O-H2O substitution with freeze-drying provides the best unquenching procedure found for in situ DNA. DNA-associated fluorescence for light microscopy can be created by moderate exposure of the specimen in the electron microscope.

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifidable in situ via the fluorescence excited by the focused electron beam of a canning electron microscope. A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam. Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules. Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues. The theoretical resolution limit for localization of a particular molecular species -- about 20 A--is determined by the known maximum distance for molecular excitation by fast electrons. Drect extapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A. Fluorescence is strongly quenched by residual water in the specimen. Similar quenching is produced by some macromolecular associations and so may serve to localize such associations.