Scientific grade CCDs operated in cooled, slow-scan cameras offer numerous advantages over single-channel and other multichannel detectors in analytical spectroscopy. Although these advantages have long been recognized and exploited, the applicability of CCDs to analytical spectroscopy has recently been expanded by the development of devices with unique capabilities. Specifically, analytical plasma emission spectroscopy requires a multichannel detector with a very large number of pixels and which has very high resistance to blooming. Analytical Raman spectroscopy is currently undergoing a push to use longer laser excitation wavelengths, and at these longer wavelengths high sensitivity becomes an even greater concern. Two new CCDs and their application in these two fields of analytical spectroscopy are described. Preliminary results obtained with a large format antiblooming CCD used with a custom-built echelle spectrometer are presented, and the capabilities of a red-response- enhanced CCD for tunable near-IR Raman spectroscopy are compared with Fourier transform Raman spectroscopy.
This paper outlines briefly some of the more important developments of the last two decades which have improved the basic performance of the microscope with respect to lateral resolution and image contrast. Such changes are due to new optical technology, digital image recording and processing, and the introduction of multimode capabilities. The introduction of laser scanning techniques using the confocal principle, together with the extension of imaging capabilities outside the visible spectrum have further extended the information available from the optical microscope. It seems probable that the use of techniques such as near-field microscopy, by breaking away from diffraction-limited imaging, will increase the spatial resolution of the optical microscope so that it approximates to that of the electron microscope. Use of improved image-capture devices and video techniques will improve temporal resolution by increasing our understanding of events which occur very rapidly or very slowly. Applying all of these changes will give use a chance to study localized chemical events as they happen in living cells in real time. It is also likely that methods of obtaining information about fine structural changes occurring in three dimensions, now just becoming practicable, will be extended so that microscopy becomes truly four-dimensional.
In the confocal scanning light microscope a specific volume is sampled during the imaging process. The physical process is explained, together with how the size of the pinholes used affects the actual size of this volume. The thus produced 3-dimensional imaging is of high quality but subject to a number of limitations. A novel (bilateral scanning) arrangement is presented which may relieve some of these. Use is made in this approach of a double sided scanning mirror element and a charge coupled device (CCD) for image collection.
The last five years have seen the rapid development and utilization of Raman Spectroscopy done with near infrared iasersL23. The major advantage of working in this spectral region is the almost total elimination fluorescence background problems normally encountered in Raman experiments utilizing visible excitation. There is, however a price to be paid. Since the Raman scattering cross-section follows a n4 law, the intensity of the Raman effect in the near infrared is approximately twenty times weaker than in the visible. This fact, coupled with the absence of good, shot noise limited detectors in the near infrared, dictates that spectral sensitivity will be low. In order to compensate for this loss in sensitivity, the obvious approach was to use a multiplexing spectrometer such as a Michelson interferometer. The performance of such a system proved to be superior to that possible using dispersive, single channel methods in the near infrared. The recent introduction of multichannel detectors, with sensitivities at wavelengths beyond one micron has changed this situation
State-of-the-art array detector technology is having a profound impact on numerous types of chemical analysis. The unique capabilities of these detectors create unprecedented opportunities in the various domains of high resolution, ultra sensitive optical spectroscopy. These array detectors, including charge coupled devices (CCDs) and charge injection devices (CIDs) have found successful application in molecular fluorescence, chemiluminescence, and atomic emission spectroscopies. The potential of these detectors for spatial and spectral imaging has been realized in a variety of applications including thin layer chromatography and flow cytometry. Applications of array detectors to these areas are discussed along with the inherently unique methods of operation such as random access integration, time delay integration, and flat fielding.
Several large-format CCDs have been designed and are in process at the Loral Aeronutronics fabrication plant. One is an edge-buttable 2048 X 2048 device that will allow a 2 X 2 array to be formed with an imaging area measuring more than 61 mm on a side and with only 400 microns dead space between arrays. Another is a 3072 X 1024 CCD with both floating diffusion and non-destructive read floating gate amplifiers. Also included are smaller arrays of 2688 X 512 and 1200 X 800 in the chords of the wafers. All of these designs were accomplished by a non-specialist scientist using AutoCAD on an inexpensive PC, a level of customer interaction with CCD manufacturing not previously available.
Biological samples have been imaged using microscopes equipped with slow-scan CCD cameras. Examples are presented of studies based on the detection of light emission signals in the form of fluorescence and phosphorescence. They include applications in the field of cell biology: (a) replication and topology of mammalian cell nuclei; (b) cytogenetic analysis of human metaphase chromosomes; and (c) time-resolved measurements of DNA-binding dyes in cells and on isolated chromosomes, as well as of mammalian cell surface antigens, using the phosphorescence of acridine orange and fluorescence resonance energy transfer of labeled lectins, respectively.
Astronomers have been the leading exploiters of CCDs, other solid state devices as well as sensitive photographic emulsions for optical imaging. Techniques associated with the use of these devices for observational measurements and the processing of data for applications is described. Practical aspects of the use of CCDs and of large digital data sets encountered in astronomy are likely to be met in other scientific applications. The experience in astronomy is therefore a source of solutions for the laboratory scientist.
Charge-coupled devices (CCDs) have become extremely important detectors for the entire astronomical community. We discuss their properties in relation to astronomical imaging and spectroscopy. We also consider some of the improvements we hope to see to further their use in astronomy and other scientific fields. These include larger area detectors and mosaics of detectors with flat and stable packaging, antireflection coatings on back illuminated devices, and extremely low read noise for spectroscopic applications. We discuss some of our research into these areas.
Many consumer products are complex physico-chemical systems, and there is a need to characterize them in the three spatial dimensions and in time. Spectroscopic imaging provides the opportunity to do this non-destructively and in-situ. New developments in detector technology, microscope optics and spectroscopics probes are beginning to generate new types of imaging experiments. Several application examples are given covering both macro- and micro-imaging.
A quantitative technique based on fluorescence microscopic detection has been developed for measurements of photosensitizer distributions in sections of tissue and cultured cells. Imaging of photosensitizer fluorescence was achieved with negligible interference from sensitizer photodegradation and tissue autofluorescence using laser excitation and detection with a highly sensitive CCD (charge-coupled device) camera system. Techniques for the measurement of sensitizer fluorescence decay parameters in living biological cells are also described.
Recent advances in two optical imaging techniques are helping scientists develop a better understanding of the development, organization and function of the cortex for sensory information processing and higher brain functions. These new clues to a better understanding of the brain are obtained as the collective activity of million of neurons is imaged simultaneously, rather than recording the individual activity of single neurons using classical electrophysiological techniques. In the first method, voltage sensitive dyes are used to image the flow of information from one cortical site to the next in real time. The second method is based on monitoring intrinsic changes in the optical properties of active brain tissue, permitting high resolution imaging of the functional architecture of cortex. These developments have only been realized as newer optical and imaging technologies are being adopted in this rapidly expanding field.
Capillary zone electrophoresis is fast becoming one of the most sensitive separation schemes for sampling complex microenvironments. A unique detection scheme is developed in which a charge-coupled device (CCD) detects laser induced fluorescence from an axially illuminated electrophoresis capillary. The fluorescence from an analyte band is measured over a several centimeter section of the capillary, greatly increasing the observation time of the fluorescently tagged band. The sensitivity of the system is in the 1-8 X 10-20 mol range for derivatized amino acids and peptides. Subattomole quantities of bag cell neuropeptides collected from the giant marine mollusk Aplysia californica can be measured.
The imaging properties of a confocal microscope depend, essentially, on the form of detector used and the lens pupil functions. We consider the use of coherent detectors and discuss an enhancement of the straight edge response by the use of a simple filter. We also show that confocal operation is achieved if a suitable optical fiber is used as a detector.
This paper will introduce a simple model for calculating the quantum efficiency of back- illuminated CCDs. The model incorporates parameters which account for what are believed to be the important features of the back surface: a surface recombination velocity, an electric field which can assist in or oppose the collection of photogenerated carriers and a field free region. Preliminary calculations of device quantum efficiency using the model agree with earlier results and indicate only moderate fields are required to enhance the quantum efficiency especially at short wavelengths.
A variety of conceptual and technological advances made during the 1980s have greatly increased the attractiveness of Raman spectroscopy for broad applications. These include fiber optic sampling, FT-Raman, CCD detectors, and single monochromators.
The Hadamard multiplexed Raman imaging microscope is described. Techniques for multispectral imaging and for contrast enhancement and optical sectioning by nearest-neighbor deblurring are discussed and illustrated. Error sources are discussed
Images of fluorescently tagged latent fingerprints were obtained using a low power source and a scientifically operated charge-coupled device (CCD) detector. The luminescence of the fingerprints is chemically enhanced with a fluorescent tag, orthophthalaldehyde. The orthophthalaldehyde undergoes a Schiff base reaction with phenyl ring containing amino acids to produce fluorescence emission at approximately equals 446 nm under UV illumination. An inexpensive, portable, low power UV source was constructed utilizing two 4-watt UV fluorescent lamps and appropriate filters. In the past, the use of filtered lamp sources resulted in an appreciable loss in sensitivity compared to laser sources. Preliminary investigations into the use of a low power tungsten filament lamp source for the excitation of NBD chloride tagged fingerprint on paper were also conducted. Sensitive detection by way of a CCD eliminates the need for the use of expensive, high power laser sources in field instruments and provides a wide range of additional advantages over photographic film.