Optical microscopy versus scanning electron microscopy in urolithiasis.

Stone analysis is incompletely done in many clinical centers. Identification of the stone component is essential for deciding future prophylaxis. X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM) still remains a distant dream for routine hospital work. It is in this context that optical microscopy is suggested as an alternate procedure. The objective of this article was to assess the utility of an optical microscope which gives magnification of up to 40x and gives clear picture of the surface of the stones. In order to authenticate the morphological analysis of urinary stones, SEM and elemental distribution analysis were performed. A total of 250 urinary stones of different compositions were collected from stone clinic, photographed, observed under an optical microscope, and optical photographs were taken at different angles. Twenty-five representative samples among these were gold sputtered to make them conductive and were fed into the SEM machine. Photographs of the samples were taken at different angles at magnifications up to 4,000. Elemental distribution analysis (EDAX) was done to confirm the composition. The observations of the two studies were compared. The different appearances of the stones under optical illuminated microscopy were mostly standardized appearances, namely bosselations of pure whewellite, spiculations of weddellite, bright yellow colored appearance of uric acid, and dirty white amorphous appearance of phosphates. SEM and EDAX gave clearer pictures and gave added confirmation of the stone composition. From the references thus obtained, it was possible to confirm the composition by studying the optical microscopic pictures. Higher magnification capacity of the SEM and the EDAX patterns are useful to give reference support for performing optical microscopy work. After standardization, routine analysis can be performed with optical microscopy. The advantage of the optical microscope is that, it is easy to use and samples can be analyzed in natural color.

Ultrastructural study of laminated urinary stone.

Several modalities of stone analysis are utilised in different laboratories. However, the treating clinician finds it hard to assess the initiation and progression of stone formation. The pathogenesis of calculogenesis still remains a mystery. The purpose of this paper is to assess the pathological mechanisms of stone nucleation and growth by observing the ultra microscopic morphology of the different layers of laminated stones; 130 fragments from 28 randomly selected laminated stones of more than 10-mm diameter were analysed. Wet chemical analysis of the stones was performed. Surface and cross-sectional morphology of the entire stones and the individual fragments was assessed using optical microscopy and images were recorded using ordinary camera. They were further analysed using FTIR for confirmation. By morphological analysis, whewellite, weddellite, uric acid, and phosphate were the main minerals identified. Mixtures of these minerals were also found. Concentric lamination, radial striation, frond formation, and amorphous pattern were the main cross-sectional morphologies obtained. The calculi analysed had differences in their outer and inner portions. This was more pronounced in stones containing predominantly whewellite and uric acid. Whewellite was the outer component in most mixed stones. Uric acid was more in the inner layers of mixed stones than the surface.

Problem in analyzing cystine stones using FTIR spectroscopy.

Cystine stones are produced by an inherited disorder of the transport of amino acid cystine that results in excess of cystine in the urine (cystinuria). Cystine calculi in urinary tract present a significant problem in patients. We have recorded that cystine calculi are very uncommon in our region. Cystine crystals are unusually identified in the urinary deposits. The problem of recognizing cystine by FTIR as a component in mixture of stones is significant. The problem is compounded by the similarity of wavelengths of cystine with that of whewellite and uric acid. The objective of this paper is to elucidate the problems of identifying cystine in stone analysis and identifying a solution to get over this deficiency. Out of 1,300 urinary stones analysed by ordinary wet chemical methods and infrared spectroscopy, 30 stone samples, which were reported to have cystine peaks in significant numbers, were selected. These samples were powdered, mixed with potassium bromide, pelletized and taken up for FTIR analysis. The wavelength patterns were scrutinized by comparing with the peaks obtained by the reference standards of cystine. Spectra were also obtained from pure cystine. Comparison of spectra with those of whewellite and uric acid was performed. Then the samples were taken for Scanning electron microscopy with elemental distribution analysis X-ray (SEM-EDAX). The samples were made conductive by gold sputtering and were fed into JEOL JSM 35 C SEM machine. Morphology was recorded by taking photographs. Further elemental distribution analysis (EDAX) was carried out to identify the elemental composition. Of the 30 samples taken up for FTIR analysis, all showed spectra identifiable with the reference peaks for cystine. However, when these peaks were compared with those of whewellite and uric acid, all the stone samples showed duplication of peaks for whewellite and uric acid and whewellite. The pure cystine spectra showed identifiable peaks are in the range of 3026, 1618.28, 1485, 846.75 cm(-1), etc. (from the standard spectrum of pure cystine). All the analysis findings were correlated with EDAX findings. On doing EDAX, we could separately find out the components present in a mixture. Three stones contained elemental pattern to fit with those of cystine. Even though it is difficult to find out the presence of cystine molecule in FTIR, it is possible to recognize it through EDAX and will be possible to confirm the presence of cystine in mixed urinary stones.

Elemental distribution analysis of urinary crystals.

Various crystals are seen in human urine. Some of them, particularly calcium oxalate dihydrate, are seen normally. Pathological crystals indicate crystal formation initiating urinary stones. Unfortunately, many of the relevant crystals are not recognized in light microscopic analysis of the urinary deposit performed in most of the clinical laboratories. Many crystals are not clearly identifiable under the ordinary light microscopy. The objective of the present study was to perform scanning electron microscopic (SEM) assessment of various urinary deposits and confirm the identity by elemental distribution analysis (EDAX). 50 samples of urinary deposits were collected from urinary stone clinic. Deposits containing significant crystalluria (more than 10 per HPF) were collected under liquid paraffin in special containers and taken up for SEM studies. The deposited crystals were retrieved with appropriate Pasteur pipettes, and placed on micropore filter paper discs. The fluid was absorbed by thicker layers of filter paper underneath and discs were fixed to brass studs. They were then gold sputtered to 100 A and examined under SEM (Jeol JSM 35C microscope). When crystals were seen, their morphology was recorded by taking photographs at different angles. At appropriate magnification, EDAX probe was pointed to the crystals under study and the wave patterns analyzed. Components of the crystals were recognized by utilizing the data. All the samples analyzed contained significant number of crystals. All samples contained more than one type of crystal. The commonest crystals encountered included calcium oxalate monohydrate (whewellite 22%), calcium oxalate dihydrate (weddellite 32%), uric acid (10%), calcium phosphates, namely, apatite (4%), brushite (6%), struvite (6%) and octocalcium phosphate (2%). The morphological appearances of urinary crystals described were correlated with the wavelengths obtained through elemental distribution analysis. Various urinary crystals that are not reported under light microscopy could be recognized by SEM-EDAX combination. EDAX is a significant tool for recognizing unknown crystals not identified by ordinary light microscopy or SEM alone.

EDAX versus FTIR in mixed stones.

Mixed stones form a significant number of all urinary stones. Accurate analysis of individual areas of stones is fraught with uncertainties. Scanning electron microscopy with elemental distribution analysis (SEM-EDAX) is a very important tool in assessing stone composition. The objective of this paper is to project the role of the combination of Fourier transform infrared (FTIR) spectroscopy and SEM-EDAX combination in achieving a total understanding of mixed stone morphology. Ten mixed urinary stones were washed and dried and the composition recognized by analysis of FTIR spectra by comparing with the spectra of pure components. Spectra for different layers were obtained. Then the stone samples were further studied by SEM-EDAX analysis. The findings of FTIR were correlated with SEM-EDAX and detailed data generated. Using SEM-EDAX, the spatial distribution of major and trace elements were studied to understand their initiation and formation. As much as 80% of the stones studied were mixtures of calcium oxalate monohydrate (whewellite) and calcium phosphate (hydroxyapatite) in various proportions. Quantitative evaluation of components was achieved through FTIR and SEM-EDAX analysis. It was possible to get an idea about the spatial distribution of molecules using SEM analysis. The composition of different areas was identified using EDAX. Analyzing with EDAX, it was possible to obtain the percentage of different elements present in a single sample. The study concludes that the most common mixed stone encountered in the study is a mixture of calcium oxalate monohydrate and calcium phosphate in a definite proportion. The combination identified not only the molecular species present in the calculus, but also the crystalline forms within chemical constituents. Using EDAX, the amount of calcium, phosphorus, oxygen and carbon present in the stone sample could be well understood.

Metabolite signature of developmental foregut cyst on in vivo and in vitro (1)H MR spectroscopy.

Foregut duplication cysts are developmental anomalies of the bronchopulmonary foregut and are common cystic lesions of the mediastinum. We describe a case of mediastinal foregut duplication cyst with in vivo (1)H MR spectroscopy on a 1.5T magnet showing a large metabolite peak at 2.02 ppm, attributable to N-acetylated compounds, in addition to a smaller peak at 1.33 ppm, considered to represent lipids. In vitro NMR spectroscopy (7.05T) of cyst fluid confirmed the presence of these peaks. In addition, a broad multiplet centered at 3.7 ppm, possibly from various protons of the hexose ring system, was also noted. Chemical analysis of the cyst fluid demonstrated the presence of N-acetylhexosamines, proteins, and lipids. Again, in vitro spectra of pure samples of N-acetylglucosamine and N-acetylgalactosamine were obtained for comparison, which better resolved the N-acetyl peak and the peaks at 3.7 ppm. The mucus secreted by respiratory epithelium and the mucous glands of the foregut cysts contains glycoproteins that have N-acetylhexosamines as components and lipid breakdown products that are thought to contribute to the observed spectrum. This information might be useful in predicting the cyst content and, in turn, the lining of the epithelium and the glandular elements.