Absorption coefficient and purine photobleaching rate in colon mucosa during resonance Raman spectroscopy at 251 nm.

In contrast to spectroscopy at longer wavelengths, typical attributes of ultraviolet resonance Raman (UVRR) spectroscopy of biologic tissue are higher absorption coefficient, mu, and higher photobleaching rate, kappa. This study was aimed at measuring mu and kappa during UVRR spectroscopy of human colon tissue at 251 nm. mu was used to estimate the penetration depth of the excitation light; kappa was used to predict the rate of signal decrease that was due to photobleaching as a function of laser fluence and tissue thickness. The fitting of the equations through description of a three-state transition model to experimental data that consisted of a purine UVRR signal gave mu = 0.0169 ? 0.0023 mum(-1) and kappa = 0.572 ? 0.168 (mJ/mum(2))(-1). kappa remained independent of power P for P < 1 mW, but higher power values resulted in a higher photobleaching rate. As predicted by the model, signal decrease that was due to photobleaching was slower as sample thickness was increased.

Optical scatter imaging: subcellular morphometry in situ with Fourier filtering.

We demonstrate a quantitative optical scatter imaging (OSI) technique, based on Fourier filtering, for detecting alterations in the size of particles with wavelength-scale dimensions. We generate our scatter image by taking the ratio of images collected at high and low numerical aperture in central dark-field microscopy. Such an image spatially encodes the ratio of wide to narrow angle scatter and hence provides a measure of local particle size. We validated OSI on sphere suspensions and live cells. In live cells, OSI revealed biochemically induced morphological changes that were not apparent in unprocessed differential interference contrast images. Unlike high-resolution imaging methods, OSI can provide size information for particles smaller than the camera's spatial resolution.

Analysis of nucleotides and aromatic amino acids in normal and neoplastic colon mucosa by ultraviolet resonance raman spectroscopy.

The objective of this study was to explore the potential of using ultraviolet resonance Raman (UVRR) spectroscopy to analyze normal and neoplastic colon tissue. Ultraviolet light at 251 nm, generated from the third harmonic of a Titanium:Sapphire laser, was used to irradiate the surfaces of surgically resected human colon specimens from six patients, five clinically diagnosed with adenocarcinoma, and one with familial adenomatous polyposis. All grossly neoplastic samples found to contain mucosal dysplasia or invasive adenocarcinoma upon histologic evaluation, were analyzed in parallel with normal tissue obtained from the same specimen and located at least 1 cm away from grossly neoplastic tissue. The colon spectra were modeled as a linear combination of nucleotide, aromatic amino acid, and lipid lineshapes, using chemical standards as a reference. Nucleotide and amino acid contributions to the UVRR spectra were quantified by a least squares minimization method. The least squares minimization spectral model was verified in aqueous solutions, where relative concentrations of free nucleotides and DNA were quantified with < 10% error. Of the 11 neoplastic samples studied from the 6 specimens, 10 showed either a lower amino acid/nucleotide ratio, a lower level of adenyl (A) signal, or both when compared with their normal counterpart. Lower amino acid/nucleotide ratio was present in five of six samples containing only dysplasia, and three of the five samples containing invasive adenocarcinoma. Lower A was present in all five samples containing invasive cancer, and in three of the six samples containing only dysplasia. This lower level of A corroborates previously published biochemistry work showing a lower level of total adenylates in tumor homogenates compared with normal tissue. Our data indicate that surface UVRR may provide unique information about site-to-site changes in cellular metabolites during colon carcinogenesis.

Correlation between synthetic activity and glycosaminoglycan concentration in epiphyseal cartilage raises questions about the regulatory role of interstitial pH.

Current data provide compelling evidence that the pH of the interstitial fluid of cartilage is an important determinant of the metabolic activity of chondrocytes, and this has served as the basis for a mechanistic proposal whereby chondrocytes could sense mechanical compression. The objective of the current study was to test this hypothesis further by examining biosynthetic activity in cartilage as a function of glycosaminoglycan content, which is the major determinant of interstitial pH. On the basis of previous data, increased biosynthetic activity would be anticipated to correlate with a decreased glycosaminoglycan content and an elevated interstitial pH. In contrast to our expectations, we found that the biosynthetic activity (monitored by measurement of incorporation of sulfate and proline) was positively correlated with the glycosaminoglycan content of tissue. These results raise doubt as to whether interstitial pH provides a dominant mechanism for controlling the metabolism of chondrocytes.

Time-dependent changes in the response of cartilage to static compression suggest interstitial pH is not the only signaling mechanism.

The goal of the present study was to reexamine the role of interstitial pH in regulating the biosynthetic rate in cartilage tissue by addressing two research questions: (a) Do small, short-term changes in interstitial pH, induced independently by two different mechanisms (namely, by controlling the pH of the medium or by mechanical compression), result in biosynthetic rates commensurate with those expected from the "natural" relationship between interstitial pH and biosynthesis? and (b) Are the effects of changes in the pH of the medium or in compression the same for short-term (14-hour) and long-term (60-hour) exposures? Biosynthetic rates were estimated from incorporation of sulfate and proline into explants of bovine epiphyseal cartilage during the final 14 hours of culture. These rates decreased with decreasing pH of the medium, with increasing compression, and with decreasing native glycosaminoglycan content; or, expressed in terms of interstitial pH, acidification induced by compression or by lowering the pH of the medium resulted in a decreased biosynthetic rate, whereas interstitial acidification effected by increasing glycosaminoglycan content enhanced it. When the time for which tissue was exposed to changes in the pH of the medium was increased from 14 to 60 hours, the relationship between the biosynthetic rate and the pH remained constant whereas the relationship between the biosynthetic rate and compression was reversed. These data suggest that the transduction mechanisms underlying the response to pH of the medium and compression differ and that some adaptation or stimulation by modest levels of compression can occur with longer exposures. Interstitial pH is not the sole determinant of biosynthesis, and it cannot really account for the long-term response of cartilage tissue to static compression.