An important function of renal epithelial cells is the reabsorption of volume, Na+ and bicarbonate (HCO3) from the filtrate. About 85% of the filtrate’s HCO3−− and 70% of the Na+ are being reabsorbed in the proximal tubule segment of the nephron. As a result, these cells are being continuously subject to large fluxes of Na+ and base through the cell. Renal medullary cells, for instance, are being subject to large fluctuations in osmolality of the extracellular fluid. Transient imbalances in fluid, base and Na+ flow through the cell can cause substantial alterations in cellular volume, pH and electrolytes.

The regulation of transepithelial transport on one hand and of cellular volume-, pH- and electrolyte-homeostasis on the other hand, is of a major physiological interest. Angiotensin II (Ang II) is a powerful regulator of Na+and HCO3− transport in the proximal tubule. The involvement of ion channels and electrogenic transporters in cellular homeostasis, has been studied by many laboratories using conventional elecrophysiological techniques to measure the resulting changes in membrane potential and ionic currents.

A rapid Ca++ signal is known to be the main trigger for exocytosis in excitable cells. However, its mode of action is unknown. Recently, it has become clear that the spatial distribution of a Ca++ stimulus is important for exocytosis. To investigate this question we have developed a novel instrument capable of imaging Ca++ gradients in patch clamped cells. We have equipped a standard fluorescence microscope with a CCD camera and an image processing station. This combination can generate a thin section view of the fluorescence of a single cell. We have equipped this microscope with a pulsed laser illumination system. The distribution of intracellular calcium can be obtained by exciting the Ca++ indicator dye (e.g., rhod-2) with a brief laser pulse [300 ns long at 525 nm ], then an image can be formed with the light emitted by the dye. by synchronizing the laser pulse with a depolarizing stimulus in a patch-clamped chromaffin cell loaded with the fluorescent Ca++ indicator rhod-2, we could easily obtain snapshots of the Ca++ distribution at known times after a stimulus.

The intrinsic fluorescence achieved without dyes (autofluorescence) of bladder tissue, benign mucosa and the various stages of transitional cell carcinoma (TCC), is being characterized. D'Hallewin, et al., and Koenig, et al., are testing autofluorescence spectroscopy for detecting bladder carcinoma using an optical fiber probe inserted down the auxiliary channel of an endoscope. These studies do not include fluorescent microscopy to directly examine the fluorescing structures (fluorophores) within the tissue. Therefore, frozen unstained sections are being imaged on a fluorescence microscope to identify and reveal the morphology and location of the fluorophores. Tissue biopsies taken at endoscopy are immediately frozen in liquid nitrogen. Later, the tissue is cut into 5 μm sections. Serial sections are alternately imaged frozen on the fluorescence microscope or stained with hematoxylin-and-eosin (H&E). The H&E stained slides are reviewed by a pathologist.

A 75-W xenon arc lamp fitted with a 380-nm narrow-band interference filter is coupled to microscope slides on the stage of an Olympus MT-2 inverted microscope.

Comparative Genomic Hybridization (CGH) is a quantitative technique for determining relative copy numbers of DNA sequences in whole-genomic test samples. In conventional CGH, test DNA labelled with one fluorochrome and reference DNA labelled with a spectrally different fluorochrome are hybridized to metaphase chromosomes. by measuring the ratio of intensities of the two fluorochromes, a relative copy number of sequences in the test DNA can be calculated for each point along each chromosome. While this approach is very useful for rapid surveying of the entire test genome, the spatial and dynamic resolution are compromised by the dense packing of DNA in the metaphase state. CGHa (array-based CGH) uses spots of cloned DNA arrayed onto a microscope slide which represent the entire genome or interesting sections thereof. Spatial resolution and dynamic range are now only limited by the size of the clones used. See the abstract by Pinkel et al. for more detail.

We designed an imaging system for analyzing fluorescence signals from micro-arrays containing targets on the order of 100μm in diameter spaced at similar intervals.

This communication presents a summary of the most recent developments in microRaman instrumentation - point analysis and imaging techniques- including the use of confocal optics and line illumination, as now available in certain commercial instruments.

The major development in micro-Raman spectroscopic instrumentation during the last few years is based on the extension of the principles of confocal microscopy. Applied to Raman microprobing the confocal technique provides an efficient way to obtain interference-free Raman spectra, as well as two-or three-dimensional, selective images of small specimens embedded within strong scatterers or fluorescent transparent media. As it is well known, the confocal configuration benefits from the use of spatial filtering by optically conjugated pinhole diaphragms. Such a system isolates the light originating from a small region of the sample coincident with the diffraction-limited, focused laser spot, and efficiently eliminates the contributions from out-of-focus zones.

A new concept of Raman imaging that benefits from the advantages of confocal microscopy has been developed in this laboratory.

The near-field scanning optical microscope, or NSOM, provides spectroscopists with resolution beneath the diffraction limit. In the NSOM, an optical aperture smaller than the wavelength λ of the probe radiation is scanned in the near-field of a sample. Pixels are serially gathered and then constituted as a computer-generated image. Spectroscopic NSOM investigations demonstrating sub-λ, resolution include studies of photoluminescence, Raman spectroscopy, and single molecule fluorescence. Results of nano-Raman spectroscopy on semiconducting Rb-doped KTP are shown in figure 1. Figure la is a topographic image of the sample showing a square Rb-doped region in an otherwise undoped sample. Figure lc is a NSOM region of the corner of the doped region, and figure lb is an image of the same region taken within a Raman line. While these data do provide sub-λ spectroscopic resolution and other interesting features, the weak signal provided by current NSOM technologies and the low quantum efficiency of the Raman effect necessitated development of a very low-drift microscope and inconveniently long collection times.

Chemical imaging of complex multi-component materials has important potential for the analyst in many fields of research. Raman imaging is of particular interest for several reasons. The Raman spectra contain detailed information on chemical species and crystalline phase. Because the Raman effect is excited by optical radiation, the spatial resolution, which is proportional to the wavelength of the light, is better than 1 μm. and with near field optical techniques currently under development, there is potential for even higher spatial resolution in the chemical image.

The methods used to produce an image fall into essentially two categories - global imaging and confocal mapping. When creating global images, a large area of the sample is bathed in laser light. The light scattered by the sample is filtered to select a Raman band, and then that light is used to create an image of the sample on a two-dimensional detector.

Confocal Raman microscopy is an invaluable tool for non-invasive three-dimensional (3-D) analysis and visualization of chemically heterogeneous samples. The confocal Raman microscope (CRM) combines focused laser illumination with spatially filtered detection to define in the sample a localized volume element (voxel) from which scattered or reflected light is detected. Attenuation of light from outside the voxel by the pinhole spatial filter increases image contrast and results in voxel sizes typically <10−18 m3. Moving the specimen relative to the voxel and recording the scattered light Raman spectrum at each scan position generates a Raman spectrally-encoded 3-D sample image for non-invasive in situ differentiation between optically similar yet chemically distinct materials.

The motivation for the present work stems from our application of continuum modeling techniques as the mathematical framework for development of physiologically realistic biosystem models. A Type 2 correlation-based confocal scanning laser microscope (CSLM) integrated with a high performance, parallel-drive micro-motion robot (MR-1) was built for spatially-distributed acquisition of optical and mechanical data within a < 1 mm3 workspace volume.

A confocal scanning Raman microscope was constructed for spectroscopy and microscopy of biological samples (Fig. 1). The microscope contains an illumination system in which a focused krypton laser beam, of which the 647.1 nm line is used to reduce damage, is scanned over the sample using a scanning mirror which moves around two orthogonal axes. Raman scattered light is collected by a water immersion objective which directs the light on the scanning mirror. Spectral analysis takes place in a monochromator with two exit ports: one for spectroscopical purposes the other one for imaging. An image is made by scanning the light, present in the Raman band passed by the monochromator, over a CCD using a second scanning mirror which moves synchronously with the first mirror. The spatial resolution is of the order of 0.3 × 0.3 × 1.2 μm3 (x,y,z).

With this microscope we studied the following samples:

In this paper we report the continuing development of a novel fiber-optic coupled confocal Raman microscope which can offer spectral resolutions up to 0.6 cm−1 per pixel, spatial resolutions of 1 micron or less with axial resolutions of 2-3 microns. The system is based around a compact base unit which comprises a compact solid-state laser, a proprietary f/1.8 imaging spectrograph, and a TE-cooled CCD detector operating at −70°C.

In Figure 1 a schematic of the fiber coupled microscope is shown. The microscope includes a integrated holographic filter module to prevent silica Raman generated in the excitation fiber from reaching the sample and to reject radiation at the excitation wavelength from being returned to the spectrograph via the collection fiber(s). The architecture has been designed to allow both fluorescence imaging and Raman microspectroscopy to be accomplished without removing the sample. The figure also depicts the positions of linear polarizers which can be used to collect polarized Raman spectra from small particles or thin polymer laminate samples.

The development of scanning near-field optical microscopy has provided a submicron optical tool which has important applications in both fundamental studies and in device fabrication and analysis. The strength of the technique lies not only in its resolution of approximately 50nm, which is an order of magnitude improvement on diffractive optical techniques, but also in its ability to allow highly developed optical spectroscopies to be used for chemical analysis at this resolution. We have particular interest in the application of near-field probes to high spatial resolution laser spectroscopy in a range of organic, biological and inorganic systems. In this paper we shall describe the development of instrumentation for luminescence, Raman and time resolved spectroscopy in air and under fluids and will present the results of studies of phase separated polymer mixtures, organic crystals and semiconductor materials.

The current scanning near-field optical microscope is based around a tapered optical fibre, at the end of which a submicron aperture has been defined by a metal coating.

The Papanicolaou (Pap) smear is the technique used to screen for abnormalities in cervical cells that may be related to cervical cancer and other problems. A number of research groups have reported results that indicate the infrared or NIR spectroscopy can be used to differentiate between samples of normal cervical cells and abnormal cells.

In this paper we will describe studies performed on cervical cell samples with an infrared microscope and a motorized x-y stage. This configuration allowed us to “Map” the sample in 100 micron increments, where an infrared spectrum is acquired at each point. This integrated system also allowed chemometric algorithms developed with the Nicolet TQ Analyst software to be automatically applied to the spectral data. Samples of cervical cells were obtained from patients using the standard procedure. Part of the sample was used to prepare the Pap smear slide and the remainder was placed in a saline solution and centrifuged for several minutes. The cell suspension was then applied to a Barium Fluoride 5mm X 5mm window and dried.

Accurate and nondestructive measurement of different lipoproteins simultaneously in carotid plaque in stroke patients using near-infrared (IR) imaging spectrometry seems possible for research. The lipoprotein composition of the plaque appears to have an impact on the outcome of the disease process. Carotid atherosclerosis without associated thrombosis is frequently a benign disease that is asymptomatic, although TIAs may be present in other cases. Many patients with carotid atherosclerosis can be treated surgically by endarterectomy with high initial success and favorable long-term prognosis. The acute manifestation of carotid atherosclerosis - stroke - arises when thrombus or ulceration develop. This potentially life-threatening complication probably develops at the site of plaque fissure or rupture. Recent research by others indicates that it is not the severity of stenosis (plaque volume) that determines the outcome: it is the type of stenosis (plaque chemical composition) and the extent of collateral growth.

Infrared spectroscopy has been used to probe a variety of biological systems including for example, the determination of diseased states and the investigation of foreign inclusions in biologicals. The technique generates qualitative and quantitative information on the structure and dynamics of samples, including lipids, proteins, and non-biological constituents. The coupling of imaging modalities with spectroscopic techniques adds a new dimension to sample analysis in both the spectroscopic and spatial domains. Using a spectroscopic imaging system that incorporates a step-scan interferometer, microscope, and infrared sensitive arrays, we have investigated a variety of biological samples. This seamless combination of spectroscopy for molecular analysis with the power of visualization generates chemically specific images while simultaneously obtaining high resolution spectra for each detector pixel. The spatial resolution of the images approaches the diffraction limit for mid-infrared wavelengths, while the spectral resolution is determined by the interferometer and can be 4 cm−1 or higher.

Oral squamous cell carcinoma (SCC) positive biopsies from patients who underwent excision procedures by micrographical surgery were analyzed by FT-IR microspectroscopy in order to examine a specific fine structure of abnormal epithelial growth - the so-called keratin pearls. Taken from the oral cavity of 15 SCC positive patients, all biopsies indicated poorly or well-differentiated cancer containing smaller and larger pearls. In some cases only a few pearls were present; in others many of various sizes.

After surgery the biopsies were rapidly frozen in liquid nitrogen and stored at -80°C before thin sectioning the tissue for infrared measurements. The frozen tissue was then sliced into 8 μm thin sections and dried on an infrared-transparent Bal2window. For comparison one slice of tissue was always used for hematoxylin and eosin staining by drying it on a standard microscope slide. This procedure creates a series of thin sections on BaF2 and glass following an alternate pattern of one 8 μm section for IR microspectroscopy and the next 8 μm one for microscopy.

We have constructed a new UV Raman microspectrometer designed around an Olympus microscope, a single spectrograph and an intensified CCD detector (Fig. 1). We utilize CW excitation from either an intracavity frequency doubled Ar+ laser (257, 244, 229 nm) or a Kr+ laser (206 nm). We optimized the throughput by utilizing specially prepared dielectric coated Rayleigh rejection filters.

In one application we used this instrument to speciate and determine the spatial distribution of non diamond carbon species in CVD diamond samples (Fig. 2). We find that these non diamond carbon species are localized in the interstitial areas between diamond crystals.

In another application we demonstrated the utility of UV Raman microspectroscopy for the rapid, incisive and non-destructive characterization of meteorites and interplanetary dust particles (IDP). In addition to probing the structure and distribution of predominant mineral matrices of these materials, UV excitation enables us to the characterize the small but significant carbonaceous components included within these samples.

Several years ago this laboratory reported on the effects of stray light in an infrared microscope and its consequences on the spatial resolution achievable in a typical line scan or mapping experiment of a polymer film. In this study it was demonstrated that unless the size of the sampled area was greater than twice the wavelength of light employed for the analysis, a significant amount of stray light from near neighbors was contained in the sampled areas. Further, this generalization is only valid for sample sizes above the diffraction limit. The results of this study are summarized in Table. The ratio of the sampled area to that of the aperture size (corresponding to expected sample size) can be thought of as a degradation factor in spatial resolution. In this earlier study the sample geometry was chosen to minimize stray light effects arising from the sample size and shape.

The optical and mechanical performance of polymer articles is often very strongly influenced by the crystallinity and degree of molecular orientation in the polymer. It is therefore important to have available techniques for measuring and mapping these parameters over article surfaces and through wall thicknesses, on scales from centimetres to microns. It is also desirable that these techniques do not in themselves perturb the morphology of the polymer, for example by introducing excess orientation or crystallinity through sectioning.

In this paper we will show how Raman microscopy can provided a useful approach for mapping polymer crystallinity on the micron scale, particularly when coupled with multivariate calibration techniques for data analysis. The acquisition and treatment of data to obtain crystallinity information will be described in detail. We will also describe the development and application of polarised FTIR specular reflectance and attenuated total reflection (ATR) microbeam techniques for quantifying and mapping surface orientation of polymer articles.

Thermoplastic polymer/liquid crystal systems have found application in the generation of display devices known as thermoplastic, polymer dispersed liquid crystals (PDLCs). These systems take advantage of the beneficial properties of both components to generate a device that has unique optical properties. The liquid crystal is dielectric and responds to an electric field. The polymer confines the liquid crystal so that the cells are closed. The two components are melted together until they are miscible. At lower temperatures, the two components phase separate. The liquid crystal component is the minor phase and takes the form of many tiny droplets contained within the major-phase, polymer matrix. An application of an electric field across these systems causes the liquid crystal within the droplets to align with the field. The systems are engineered such that when this alignment occurs there is no refractive index difference between the liquid crystal in the droplets and the polymer matrix, thus, the cells appear optically transparent. When there is no field applied, the liquid crystals in each droplet are aligned without respect to a general direction according to the surface energetics of each droplet/polymer interface. When this is the case, there is a refractive index mismatch between the droplets and the polymer and the cells are opaque. Research of these systems is aimed at improving the optical properties in order to facilitate the manufacturing of improved devices utilizing this technology. Because these systems are generated by a diffusion-controlled, phase separation process, understanding the relevant parameters, particularly the diffusion coefficients, should enable the manufacturing processes of these systems to be controlled more efficiently, generating improved optical properties.

Materials characterization is the primary application of macro- and micro-Raman spectroscopy in our laboratory. Specifically, we wish to correlate chemical bonding and short to long range translational symmetry (including amorphous, highly oriented, polycrystalline, and single crystal materials) to physical, optical and electronic properties of materials and devices. Raman spectroscopy is particularly useful in this capacity because of its origin in the vibrational motions of chemically bonded atoms and its dependence upon crystal symmetry through the polarization selection rules. Furthermore, the high spatial resolution and non-destructive nature of micro-Raman spectroscopy make it ideal for in situcharacterization of electronic and photonic devices. We will present results of materials characterization studies, performed using macro- and micro-Raman spectroscopy, of electronic and photonic devices. In addition, we will discuss how the Raman polarization selection rules can be advantageously applied to device characterization.

A primary area of investigation involves the study of ion-implanted and annealed Si by Raman spectroscopy.