Author Correction: Measuring light scattering and absorption in corals with Inverse Spectroscopic Optical Coherence Tomography (ISOCT): a new tool for non-invasive monitoring.

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Measuring light scattering and absorption in corals with Inverse Spectroscopic Optical Coherence Tomography (ISOCT): a new tool for non-invasive monitoring.

The success of reef-building corals for >200 million years has been dependent on the mutualistic interaction between the coral host and its photosynthetic endosymbiont dinoflagellates (family Symbiodiniaceae) that supply the coral host with nutrients and energy for growth and calcification. While multiple light scattering in coral tissue and skeleton significantly enhance the light microenvironment for Symbiodiniaceae, the mechanisms of light propagation in tissue and skeleton remain largely unknown due to a lack of technologies to measure the intrinsic optical properties of both compartments in live corals. Here we introduce ISOCT (inverse spectroscopic optical coherence tomography), a non-invasive approach to measure optical properties and three-dimensional morphology of living corals at micron- and nano-length scales, respectively, which are involved in the control of light propagation. ISOCT enables measurements of optical properties in the visible range and thus allows for characterization of the density of light harvesting pigments in coral. We used ISOCT to characterize the optical scattering coefficient (µs) of the coral skeleton and chlorophyll a concentration of live coral tissue. ISOCT further characterized the overall micro- and nano-morphology of live tissue by measuring differences in the sub-micron spatial mass density distribution (D) that vary throughout the tissue and skeleton and give rise to light scattering, and this enabled estimates of the spatial directionality of light scattering, i.e., the anisotropy coefficient, g. Thus, ISOCT enables imaging of coral nanoscale structures and allows for quantifying light scattering and pigment absorption in live corals. ISOCT could thus be developed into an important tool for rapid, non-invasive monitoring of coral health, growth and photophysiology with unprecedented spatial resolution.

The Global Relationship between Chromatin Physical Topology, Fractal Structure, and Gene Expression.

Most of what we know about gene transcription comes from the view of cells as molecular machines: focusing on the role of molecular modifications to the proteins carrying out transcriptional reactions at a loci-by-loci basis. This view ignores a critical reality: biological reactions do not happen in an empty space, but in a highly complex, interrelated, and dense nanoenvironment that profoundly influences chemical interactions. We explored the relationship between the physical nanoenvironment of chromatin and gene transcription in vitro. We analytically show that changes in the fractal dimension, D, of chromatin correspond to simultaneous increases in chromatin accessibility and compaction heterogeneity. Using these predictions, we demonstrate experimentally that nanoscopic changes to chromatin D within thirty minutes correlate with concomitant enhancement and suppression of transcription. Further, we show that the increased heterogeneity of physical structure of chromatin due to increase in fractal dimension correlates with increased heterogeneity of gene networks. These findings indicate that the higher order folding of chromatin topology may act as a molecular-pathway independent code regulating global patterns of gene expression. Since physical organization of chromatin is frequently altered in oncogenesis, this work provides evidence pairing molecular function to physical structure for processes frequently altered during tumorigenesis.

Procedures for risk-stratification of lung cancer using buccal nanocytology.

Lung cancer is the leading cause of cancer deaths in the U.S. with survival dramatically depending on stage at diagnosis. We had earlier reported that nanocytology of buccal cells can accurately risk-stratify smokers for the presence of early and late-stage lung cancer. To translate the technique into clinical practice, standardization of operating procedures is necessary to consistently yield precise and repeatable results. Here, we develop and validate simple, robust, and easily implementable procedures for specimen collection, processing, etc. in addition to a commercially-viable instrument prototype. Results of this work enable translation of the technology from academic lab to physicians' office.

Interferometric spectroscopy of scattered light can quantify the statistics of subdiffractional refractive-index fluctuations.

Despite major importance in physics, biology, and other sciences, the optical sensing of nanoscale structures in the far zone remains an open problem due to the fundamental diffraction limit of resolution. We establish that the expected value of spectral variance (Σ[over ˜](2)) of a far-field, diffraction-limited microscope image can quantify the refractive-index fluctuations of a label-free, weakly scattering sample at subdiffraction length scales. We report the general expression of Σ[over ˜] for an arbitrary refractive-index distribution. For an exponential refractive-index spatial correlation, we obtain a closed-form solution of Σ[over ˜] that is in excellent agreement with three-dimensional finite-difference time-domain solutions of Maxwell's equations. Sensing complex inhomogeneous media at the nanoscale can benefit fields from material science to medical diagnostics.

Targeted alteration of real and imaginary refractive index of biological cells by histological staining.

Various staining techniques are commonly used in biomedical research to investigate cellular morphology. By inducing absorption of light, staining dyes change the intracellular refractive index due to the Kramers-Kronig relationship. We present a method for creating 2D maps of real and imaginary refractive indices of stained biological cells using their thickness and absorptance. We validate our technique on dyed polystyrene microspheres and quantify the alteration in refractive index of stained biological cells. We reveal that specific staining of individual organelles can increase their scattering cross-section by orders of magnitudes, implying a major impact in the field of biophotonics.

Low-coherence enhanced backscattering and its applications.

The phenomenon of enhanced backscattering (EBS) of light, also known as coherent backscattering, has been the object of intensive investigation in non-biological media over the last two decades. However, there have been only a few attempts to explore EBS for tissue characterization and diagnosis. We have recently made progress in the EBS measurements in tissue by taking advantage of low spatial coherence illumination, which has led us to the development of low-coherence enhanced backscattering (LEBS) as a technique to characterize living tissue. In this paper, we review the current state of research on LEBS. In particular, we show that LEBS spectroscopy enables detection of early microarchitectural changes in tissue associated with carcinogenesis prior to the development of histologically-detectable alterations as well as any other known markers of neoplasia. Thus, LEBS may offer insights into initial events in carcinogenesis.

Increased microvascular blood content is an early event in colon carcinogenesis.

BACKGROUND: Increased premalignant epithelial microvascular blood content is a common theme in neoplastic transformation; however, demonstration of this phenomenon in colon carcinogenesis has been stymied by methodological limitations. Our group has recently developed a novel optics technology, four dimensional elastic light scattering fingerprinting (4D-ELF), which allows examination of the colonic mucosal architecture with unprecedented accuracy. In this study, we utilised 4D-ELF to probe the preneoplastic colonic microvasculature. METHODS: Colonic mucosal blood content was assessed by 4D-ELF at serial preneoplastic time points from azoxymethane (AOM) treated Fisher 344 rats and age matched control animals. We also examined the pretumorigenic intestinal mucosa of the MIN mouse, and compared with wild-type mice. Finally, in a pilot study, we examined superficial blood content from the endoscopically normal mid transverse colon in 37 patients undergoing screening colonoscopy. RESULTS: In the AOM treated rat model, augmentation of superficial mucosal and total mucosal/superficial submucosal blood supply preceded the appearance of aberrant crypt foci (ACF) and temporally and spatially correlated with future ACF occurrence. These findings were replicated in MIN mice. The 4D-ELF based results were corroborated with immunoblot analysis for haemoglobin on mucosal scrapings from AOM treated rats. Moreover, 4D-ELF analysis of normal human colonic mucosa indicated that there was a threefold increase in superficial blood in patients who harboured advanced adenomas. CONCLUSION: We report, for the first time, that blood content is increased in the colonic microvasculature at the earliest stages of colon carcinogenesis. These findings may provide novel insights into early biological events in colorectal carcinogenesis and have potential applicability for screening.

Imaging human epithelial properties with polarized light-scattering spectroscopy.

Biomedical imaging with light-scattering spectroscopy (LSS) is a novel optical technology developed to probe the structure of living epithelial cells in situ without need for tissue removal. LSS makes it possible to distinguish between single backscattering from epithelial-cell nuclei and multiply scattered light. The spectrum of the single backscattering component is further analyzed to provide quantitative information about the epithelial-cell nuclei such as nuclear size, degree of pleomorphism, degree of hyperchromasia and amount of chromatin. LSS imaging allows mapping these histological properties over wide areas of epithelial lining. Because nuclear enlargement, pleomorphism and hyperchromasia are principal features of nuclear atypia associated with precancerous and cancerous changes in virtually all epithelia, LSS imaging can be used to detect precancerous lesions in optically accessible organs.

Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett's esophagus.

BACKGROUND & AIMS: The aim of this study was to assess the potential of 3 spectroscopic techniques (fluorescence, reflectance, and light-scattering spectroscopy) individually and in combination, for evaluating low- and high-grade dysplasia in patients with Barrett's esophagus (BE). METHODS: Fluorescence spectra at 11 excitation wavelengths and a reflectance spectrum were acquired in approximately 1 second from each site before biopsy using an optical fiber probe. The measured fluorescence spectra were combined with the reflectance spectra to extract the intrinsic tissue fluorescence. The reflectance spectra provided morphologic information about the bulk tissue, whereas light-scattering spectroscopy was used to determine cell nuclear crowding and enlargement in Barrett's epithelium. RESULTS: Significant differences were observed between dysplastic and nondysplastic BE in terms of intrinsic fluorescence, bulk scattering properties, and levels of epithelial cell nuclear crowding and enlargement. The combination of all 3 techniques resulted in superior sensitivity and specificity for separating high-grade from non-high-grade and dysplastic from nondysplastic epithelium. CONCLUSIONS: Intrinsic fluorescence, reflectance, and light-scattering spectroscopies provide complementary information about biochemical and morphologic changes that occur during the development of dysplasia. The combination of these techniques (Tri-Modal Spectroscopy) can serve as an excellent tool for the evaluation of dysplasia in BE.

Endoscopic detection of dysplasia in patients with Barrett's esophagus using light-scattering spectroscopy.

BACKGROUND & AIMS: We conducted a study to assess the potential of light-scattering spectroscopy (LSS), which can measure epithelial nuclear enlargement and crowding, for in situ detection of dysplasia in patients with Barrett's esophagus. METHODS: Consecutive patients with suspected Barrett's esophagus underwent endoscopy and systematic biopsy. Before biopsy, each site was sampled by LSS using a fiberoptic probe. Diffusely reflected white light was spectrally analyzed to obtain the size distribution of cell nuclei in the mucosal layer, from which the percentage of enlarged nuclei and the degree of crowding were determined. Dysplasia was assigned if more than 30% of the nuclei exceeded 10 microm and the histologic findings compared with those of 4 pathologists blinded to the light-scattering assessment. The data were then retrospectively analyzed to further explore the diagnostic potential of LSS. RESULTS: Seventy-six sites from 13 patients were sampled. All abnormal sites and a random sample of nondysplastic sites were reviewed by the pathologists. The average diagnoses were 4 sites from 4 different patients as high-grade dysplasia (HGD), 8 sites from 5 different patients as low-grade dysplasia (LGD), 12 as indefinite for dysplasia, and 52 as nondysplastic Barrett's. The sensitivity and specificity of LSS for detecting dysplasia (either LGD or HGD) were 90% and 90%, respectively, with all HGD and 87% of LGD sites correctly classified. Decision algorithms using both nuclear enlargement and crowding further improved diagnostic accuracy, and accurately classified samples into the 4 histologic categories. CONCLUSIONS: LSS can reliably detect LGD and HGD in patients with Barrett's esophagus.

Diffuse reflectance spectroscopy of human adenomatous colon polyps in vivo.

Diffuse reflectance spectra were collected from adenomatous colon polyps (cancer precursors) and normal colonic mucosa of patients undergoing colonoscopy. We analyzed the data by using an analytical light diffusion model, which was tested and validated on a physical tissue model composed of polystyrene beads and hemoglobin. Four parameters were obtained: hemoglobin concentration, hemoglobin oxygen saturation, effective scatterer density, and effective scatterer size. Normal and adenomatous tissue sites exhibited differences in hemoglobin concentration and, on average, in effective scatterer size, which were in general agreement with other studies that employ standard methods. These results suggest that diffuse reflectance can be used to obtain tissue information about tissue structure and composition in vivo.

Determination of dimethylated arginines in human plasma by high-performance liquid chromatography.

Using high-performance liquid chromatography (HPLC) with multigradient elution, N(G),N(G)-dimethyl-L-arginine (asymmetric-DMA, ADMA) and N(G),N'(G)-dimethyl-L-arginine (symmetric-DMA, SDMA) can be separated from human plasma samples. The dimethylarginine compounds in plasma, after extraction with a cation-exchange column, are converted to fluorescent derivatives with o-phthaldialdehyde (OPA) in an alkaline medium and the derivatives are separated simultaneously within 50 min on a reversed-phase column (Ultracarb 3 ODS(20)). The recoveries of ADMA and SDMA are over 80% and the method permits quantitative determination of dimethylated arginines at concentrations as low as 0.1 micromol/l in human plasma.

A simple, rapid, and highly reliable capsule-based 14C urea breath test for diagnosis of Helicobacter pylori infection.

BACKGROUND: Since the urea breath test (UBT) indirectly detects gastric Helicobacter pylori infection by measuring urease activity, the possibility of false-positive results due to other urease-producing bacteria cannot be excluded. Previous studies have shown that increased 14CO2 activity in early breath samples could be attributed to urea hydrolysis in the oropharynx. For that reason, reliable assessment of H. pylori status is hampered for at least 20 min after administration of a 14C-urea drink. METHODS: To overcome this problem we have developed a modified breath test in which 111kBq 14C-urea is supplied in a gelatin capsule, which prevents release of 14C before reaching the stomach. Our modified 14C UBT was evaluated in 100 healthy volunteers, and results were compared with those from enzyme-linked immunosorbent assay serology. RESULTS: The study showed a 99% concordance between the two noninvasive tests. When a biometric method for determination of cut-off values between positive and negative UBT results with the smallest possible arbitrariness was used, the calculated statistical probability of a false diagnosis was lowest in the 10-min breath sample (0.20%), and 100% sensitivity and specificity was achieved. Our capsule method was also compared with the urea drink method and was found more reliable because no overlapping in 14CO2 activity occurred between H. pylori-positive and -negative subjects, whereas conventional breath testing showed overlapping during the whole 30-min test period. Our study also showed that a fatty test meal lowers the 14CO2 excretion the first 20 min and may adversely affect the accuracy of a rapid UBT. CONCLUSIONS: Supplying the 14C-urea in a capsule obviates the problem of false-positive results in early breath samples and makes it possible to diagnose H. pylori infection with 99.8% reliability from a single 10-min breath sample, without the use of a test meal of adjustments for assumed individual CO2 production.

Accumulation of an endogenous inhibitor of nitric oxide synthase during graded hemorrhagic shock.

Asymmetric dimethylarginine (ADMA) represents an endogenous inhibitor of nitric oxide (NO) production. The production of ADMA has been shown to increase during cellular stress, e.g., hypoxia. Furthermore, ADMA has recently been reported to accumulate in plasma during terminal renal failure as a consequence of diminished urinary excretion. Since tissue hypoxia and oliguria are both characteristics of severe hemorrhagic shock, this study was performed in order to establish whether plasma concentrations of ADMA increase during hemorrhagic shock. Six pigs were subjected to graded hemorrhage (20% and 40% of the calculated blood volume), resulting in significant (P < 0.05) reductions in blood pressure and cardiac output (from 98 +/- 4 to 36 +/- 5 mm Hg and from 3.0 +/- 0.2 to 1.4 +/- 0.2 L/min, respectively). Plasma ADMA concentrations as determined by high-performance liquid chromatography (HPLC) increased from a pre-hemorrhage value of 3.4 +/- 0.3 microM to 3.9 +/- 0.4 microM (ns) and 5.2 +/- 0.4 microM (P < 0.05), respectively. The present study demonstrates that plasma ADMA concentrations increase significantly during hemorrhagic shock. Thus, inhibition of the arginine-nitric oxide pathway as a result of ADMA accumulation, may represent an additional physiological mechanism to maintain systemic blood pressure in response to acute hypovolemia.

The periplasmic dipeptide permease system transports 5-aminolevulinic acid in Escherichia coli.

In a genetic screen designed to generate Escherichia coli strains completely devoid of the heme precursor 5-aminolevulinic acid (ALA), we isolated a class of mutants which were defective for exogenous ALA uptake. The mutations, designated alu (ALA uptake), mapped to the 80-min region of the E. coli chromosome. They were complemented by a recombinant plasmid containing the dpp operon, which encodes a dipeptide permease transport system. Alu mutants displayed a severe reduction in ALA import, as did a strain with a chromosomal insertion in the first gene of the dpp operon. A recognized substrate of Dpp transport, prolyl-glycine, effectively competed with ALA for uptake. E. coli strains defective in ALA biosynthesis (hemA or hemL) require exogenous ALA to achieve wild-type growth but show limited aerobic and anaerobic growth in the absence of ALA. The presence of an alu or dpp mutation in hemA or hemL strains abolishes growth in the absence of ALA and requires increased levels of ALA for normal growth. We conclude that the alu mutations are within the dpp operon and that the dipeptide transport system mediates uptake of the important metabolite ALA.