More and more, analytical chemists are beginning to realize the microscope, that most ancient of analytical tools, has unique application in the modern laboratory. Without flashing lights and transistors, light microscopes, especially the polarizing microscope, reward the analytical chemist familiar with its use, by rapidly solving many if not most of his problems.
There has been considerable interest in recent years in devising methods that can provide direct, accurate, and real time microscopic measurements of phase objects, even when the path difference variations are large. Various approaches have used direct coherent imaging, modification of phase contrast microscopy, scanning interferometry, and holographic methods. These various approaches are reviewed and examples discussed.
The Virometer instrument completes a measurement on live or dead virus in approximately fifteen minutes at concentrations as low as 107 particles per milliliter and works with sample volumes of one microliter. Early work has shown results with virus particles in the 300 R to 1500 R size range, and at concentrations of 108 to 1012 particles per milliliter. In addition to nucleic acid type and quantity, the "Virometer" also determines single or double-strandedness of DNA. The instrument includes an Argon laser, modified light micro-scope optics, and special light detection system utilizing attenuated total reflection fluorescence, and light-scatter measurement. A special mixture of RNA and DNA-revealing fluorescent dyes is added to a sample preparation. After staining takes place, the sample preparation is observed through an optical aperture smaller than a single light wavelength yielding size information via Brownian motion, even in the presence of high background found in typical biological fluids. Additional processing determines titer and individual particle nucleic acid mass. Autocorrelation and Fourier transformation are used in specialized electronics to allow essentially real-time data reduction.
Holographic techniques are ideally suited for interferometric studies on dynamic systems and are especially valuable when adapted for use with a microscope. The holographic microscope and the method of hologram production is described as well as the basic methods for holographic interferometric microscopy (HIM).' HIM can produce interferograms in either one of two distinct ways. In the first way one obtains an interferogram of a subject by reconstruction from a doubly exposed hologram, where one of the two exposures acts as the reference wavefront. In the second way of producing an interferogram, one makes a simultaneous reconstruction from two separately recorded holograms. The use of HIM is illustrated by a description of two studies that deal with dynamic systems, one diffusion and the other a crystallization. One of the major advantages of HIM is found in the use of simple experimental technique and both studies used for illustration show that no complicated optics are required for either qualitative or quantitative data.
A new technology has been developed which allows us to directly investigate the molecular properties of matter at the microscopic level. This paper will describe the technology, discuss the most recent development in the field, and offer examples which illustrate the technology's application to several diciplines.
In the nineteen thirties, before the great Government crime laboratories were established, individual scientific workers in the Federal agencies were sometimes called upon to settle important questions that the detectives could not handle. For this purpose, it was often necessary to invent new techniques to tackle problems by new approaches. The laboratory man needed to develop imaginative ways of looking at fragmentary material that became available. This presentation will deal with three episodes involving original procedures. The procedures proved their point in the issues at hand, but the findings were never used in court so far as the author is aware. They have not been published or publicly disclosed. If a board has been sawed into three pieces of which the middle one is missing, how do you establish that the end pieces were once joined? If a method of recognizing typescript is developed that provides quick and convincing evidence of identity, what are the chances that a sophisticated forger, knowing the technique of recognition, could successfully imitate the identity? What do you do if the exhibits in a murder case are effective and relevant evidence so far as the facts are concerned, but indicate that another and critical piece of evidence is missing? Suppose that without the missing piece, nothing else has firm meaning! These three examples are chosen for presentation because, although the microscopical techniques that were used are interesting, they appear to have lain dormant for over forty years.
An A.C. interference microscope based on a technique called phase-locked interferometry has been constructed. The instrument is capable of detecting phase differences approaching X/100. It may be used for measuring surface features (roughness, curvature) or transparent phase objects such as gradient index materials, microballoons and biological samples.
A simple method of image analysis suitable for arbitrary objects under partially coherent illumination is presented and applied to phase, schlieren, and interference microscopy. While approximate in the sense that diffraction is ignored, the method permits analysis using only algebra, resulting in a mathematical image in closed form.
When we define as the image of a scanning system the values of its electronic signal that corresponds to each point of the scanned object, there is a basic equivalence between the imaging properties of such systems and those of conventional imaging systems. This equivalence which is based on fundamental physical laws, en-ables us to analyze or design a scanning system in terms of a corresponding conventional imaging system.
Holographic microscopy is a useful tool in a wide variety of applications. This paper reviews the history and theory of several holographic microscopes which have been used successfully in a variety of applications. An overview of the relevant parameters of importance in designing a holographic microscope will be presented. Recording media properties and sources of noise will be discussed as they relate to the quality of the reconstructed image. A summary of several successful applications, including design considerations, will conclude this paper.
A comprehensive examination is made of recent advanced research directions in the applications of electro-optical and holographic instrumentations and methods to atmospheric sciences problems. In addition, an over-view is given of the in-house research program for environmental and atmospheric measurements with emphasis on particulates systems. Special treatment is made of the instrument methods and applications work in the areas of laser scattering spectrometers and pulsed holography sizing systems. Selected engineering tests data on space simulation chamber programs are discussed.
In the technique of analysis known as secondary ion mass spectrometry (SIMS), a beam of energetic ions (2-40 keV) strikes a sample(pd dislodges atoms lying near the surface by the process known as sputtering, Figure 1. A small fraction (104 - 10 2) of the sputtered sample atoms is emitted in a charged state, the so-called secondary ions. Because of their electrical charge, the secondary ions can be attracted by appropriate electrical fields into a mass spectrometer which disperses the ions according to their mass-to-charge ratio. Ion detection is accomplished by either an electrical detector or a photographic plate. By varying the strength of the magnetic and/or electrical field of the mass spectrometer, a mass spectrum of the positive or negative sputtered ions in the form of intensity versus mass-to-charge ratio is obtained.
The light and electron microscopes were for a long time the two principal tools for examination and identification of small particles. Although the polarizing microscope remains the single most useful instrument for such identification, the development of scanning beam instruments has tremendously increased the particle analyst's scope. He can now in many cases, examine the ultrafine structure of a small particle without special sample pre-paration by using a scanning electron microscope. In addition, he can display the particle's image while simultaneously examining the major chemical constituents of the particle using an Energy Dispersive X-ray Detecting System. For additional insight into the particle's constitution, detailed x-ray maps showing elemental distribution within the particle are produced rapidly in a scanning electron microprobe. The presence, composition, and approx-imate thickness of surface films can also be determined in the electron microprobe. Thin films and even monolayer surface films can now be detected with the scanning Auger spectrometer or the ion microprobe analyzer. Besides identifying thin films, the ion microprobe and ion microscope can give the chemical composition of a small particle's surface as well as that of its interior. In many cases, information on the type of chemical compound in a small particle can be elicited from the mass spectra of the ion beam instruments. A recent newcomer in the ever expanding field of small particle identification is Microraman Spectrometry. The Raman spectra of small particles are unique fingerprints of chemical compounds for both inorganic and organic constituents and thus come closer to the ultimate goal: positive identification of any unknown small particle.
The need to determine and display the elemental composition and distribution of elements of small samples from a few millimeters to a few microns has been recognized for some time. However, until recently such analysis was slow and the instrumentation costly. With the advent of energy dispersive spectrometers of improved (150eV) resolution and the introduction of lower cost SEM's ($25,000) which still retain 100 Angstroms resolution or better, the technique of x-ray imaging has become much more wide spread and readily available. This paper describes the various techniques of imaging involved. Samples containing large amounts of hydrocarbons, such as biological samples, have been difficult to analyze because of the low scatter which creates high backgrounds, thereby lowering sensitivity. This problem has been overcome for the most part by two relatively new techniques, the bulk mode analyzer and the low temperature asher. These instruments, when used with the SEM/EDX system, allow one to see and analyze inorganic particles otherwise hidden in a carbon matrix as well as lower the detection limits of homogenous inorganic elements down to the low (10-50) ppm range in organic matrixes. Thereby greatly extending the analytical capa-bility of an already powerful analytical tool.
An infrared MicroImager was originally introduced at SPIE in 1972. The operation of the instrument is reviewed. The application of the MicroImager in integrated circuits thermal analysis is discussed. Quantitative data relative to actual surface temperature of integrated circuits is available from infrared observations. Thermal signatures of integrated circuits obtained from MicroImager observations provide valuable information to circuit designers, process engineers and failure analysts. Thermal signatures support theoretical conclusions regarding thermal distrihutio by presenting empirical observations.
The term "Acoustic Microscopy" refers to the visualization of detail in the submillimeter to submicron range, by means of high frequency sound waves. Sound being a mechanical phenomenon, its use in microscopy reveals details of elastic structure related to stiffness, density, viscosity, etc. Because such information is not readily available from other techniques, acoustic microscopy complements the more conventional methods. In this review I will trace the historical development of acoustic microscopy from its beginning in the early '50s to the present time. I will discuss, in some detail, the technological evolution that led from the early instruments with millimeter resolution at megahertz frequencies to the present true microscopes that operate in the gigahertz region and achieve a resolution of microns.
Acoustic microscopy is a non-destructive, non-invasive technique for revealing microstructure and detecting changes in elastic properties of samples on a microscopic scale. This technique provides information not available through other techniques, and it has broad applications to biomedical research and materials science. An acoustic microscope has been developed which makes use of a scanning laser beam as a remote sensing microphone. Acoustic and optical micrographs of a specimen are simultaneously produced on TV monitors in real time, thereby enabling dynamic activity to be studied.
Information concerning muscle mechanics has been derived largely from measurements obtained by attaching force transducers on coordinate systems to the ends of the muscle, thus the information derived is the integrated result of all the properties of all of the components of the muscle distributed along its length. The acoustic microscope is an instrument which may be applied to the study of muscle mechanics and instrumented with force transducers and quick stretch and release servos. This permits measurement of transverse properties as well as changes along the length of the muscle. There are several kinds of information which can be obtained using such instrumentation. Operating in the interference mode, the microscope can be used to measure changes in transit time through the specimen which can be related to changes in the acoustic velocity which, in turn, can be related to the adiabatic bulk modulus of elasticity. Changes in the transmission can be related to local changes in viscosity of the muscle during contraction. The microscope can also be used to measure dimensions in the x-y plane and along the z axis. Using the line-scan technique, rapid changes can be recorded. The line-scan rate is 15,750 lines per second, which is sufficient for resolving details of the contractile event.
Infrared Microscopy is useful for nondestructive evaluation of semiconductor materials and devices. At wavelengths near 1100 nm, silicon and GaAs have good transparency. Therefore, internal defect structures may be observed. Techniques of infrared microscopy and methods of contrast enhancement such as decoration, interference contrast, and fluorescence are discussed. Applications are presented including examples of precipitates in silicon, die attach bond defects, and strain patterns in epitaxial layers and completed devices.