Spectrum of the cavity-QED microlaser: strong coupling effects in the frequency pulling at off resonance.

We report the first experimental observation of the cavity-QED microlaser spectrum, specifically the unconventional frequency pulling brought by a strong atom-cavity coupling at off resonance. The pulling is enhanced quadratically by the atom-cavity coupling to result in a sensitive response to the number of pumping atoms (2.1 kHz per atom maximally). Periodic variation of the pulling due to the coherent Rabi oscillation is also observed as the number of pumping atoms is increased across multiple thresholds.

Raman spectroscopy: a real-time tool for identifying microcalcifications during stereotactic breast core needle biopsies.

Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. We present here a Raman spectroscopic tool for detecting microcalcifications in breast tissue based on their chemical composition. We collected ex vivo Raman spectra from 159 tissue sites in fresh stereotactic breast needle biopsies from 33 patients, including 54 normal sites, 75 lesions with microcalcifications and 30 lesions without microcalcifications. Application of our Raman technique resulted in a positive predictive value of 97% for detecting microcalcifications. This study shows that Raman spectroscopy has the potential to detect microcalcifications during stereotactic breast core biopsies and provide real-time feedback to radiologists, thus reducing non-diagnostic and false negative biopsies.

Observation of sub-poisson photon statistics in the cavity-QED microlaser.

We have measured the second-order correlation function of the cavity-QED microlaser output and observed a transition from photon bunching to antibunching with increasing average number of intracavity atoms. The observed correlation times and the transition from super- to sub-Poisson photon statistics can be well described by gain-loss feedback or enhanced-reduced restoring action against fluctuations in photon number in the context of a quantum microlaser theory and a photon rate equation picture. However, the theory predicts a degree of antibunching several times larger than that observed, which may indicate the inadequacy of its treatment of atomic velocity distributions.

Instrumentation for multi-modal spectroscopic diagnosis of epithelial dysplasia.

Reflectance and fluorescence spectroscopies have shown great promise for early detection of epithelial dysplasia. We have developed a clinical reflectance spectrofluorimeter for multimodal spectroscopic diagnosis of epithelial dysplasia. This clinical instrument, the FastEEM, collects white light reflectance and fluorescence excitation-emission matrices (EEM's) within a fraction of a second. In this paper we describe the FastEEM instrumentation, designed for collection of multi-modal spectroscopic data. We illustrate its performance using tissue phantoms with well defined optical properties and biochemicals of known fluorescence properties. In addition, we discuss our plans to develop a system that combines a multi-spectral imaging device for wide area surveillance with this contact probe device.

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.

Path-length-resolved dynamic light scattering: modeling the transition from single to diffusive scattering.

Dynamic light-scattering spectroscopy is used to study Brownian motion within highly scattering samples. The fluctuations of the light field that is backscattered by a suspension of polystyrene microspheres are measured as power spectra by use of low-coherence interferometry to obtain path-length resolution. The data are modeled as the sum of contributions to the detected light weighted by a Poisson probability for the number of events that each component has experienced. By analyzing the broadening of the power spectra as a function of the path length for various sizes of particles, we determine the contribution of multiple scattering to the detected signal as a function of scattering anisotropy.

Measurement of angular distributions by use of low-coherence interferometry for light-scattering spectroscopy.

We present a novel interferometer for measuring angular distributions of backscattered light. The new system exploits a low-coherence source in a modified Michelson interferometer to provide depth resolution, as in optical coherence tomography, but includes an imaging system that permits the angle of the reference field to be varied in the detector plane by simple translation of an optical element. We employ this system to examine the angular distribution of light scattered by polystyrene microspheres. The measured data indicate that size information can be recovered from angular-scattering distributions and that the coherence length of the source influences the applicability of Mie theory.

Phase-dispersion optical tomography.

We report on phase-dispersion optical tomography, a new imaging technique based on phase measurements using low-coherence interferometry. The technique simultaneously probes the target with fundamental and second-harmonic light and interferometrically measures the relative phase shift of the backscattered light fields. This phase change can arise either from reflection at an interface within a sample or from bulk refraction. We show that this highly sensitive (~5 degrees ) phase technique can complement optical coherence tomography, which measures electric field amplitude, by revealing otherwise undetectable dispersive variations in the sample.

Phase-referenced interferometer with subwavelength and subhertz sensitivity applied to the study of cell membrane dynamics.

We report a highly sensitive means of measuring cellular dynamics with a novel interferometer that can measure motional phase changes. The system is based on a modified Michelson interferometer with a composite laser beam of 1550-nm low-coherence light and 775-nm CW light. The sample is prepared on a coverslip that is highly reflective at 775 nm. By referencing the heterodyne phase of the 1550-nm light reflected from the sample to that of the 775-nm light reflected from the coverslip, small motions in the sample are detected, and motional artifacts from vibrations in the interferometer are completely eliminated. We demonstrate that the system is sensitive to motions as small as 3.6 nm and velocities as small as 1 nm/s. Using the instrument, we study transient volume changes of a few (approximately three) cells in a monolayer immersed in weakly hypotonic and hypertonic solutions.

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.

Feasibility of field-based light scattering spectroscopy.

Light scattering spectroscopy (LSS) is a new technique capable of accurately measuring the features of nuclei and other cellular organelles in situ. We present the considerations required to implement and interpret field-based detection in LSS, where the scattered electric field is detected interferometrically, and demonstrate that the technique is experimentally feasible. A theoretical formalism for modeling field-based LSS signals based on Mie scattering is presented. Phase-front uniformity is shown to play an important and novel role. Results of heterodyne experiments with polystyrene microspheres that localize LSS signals to a region about 30 microns in axial extent are reported. In addition, differences between field-based LSS and the earlier intensity-based LSS are discussed.

Optical computed tomography in a turbid medium using early arriving photons.

We employ photon migration to image absorbing objects embedded in a turbid medium. For improved resolution, we use early arriving photons (a few hundred picoseconds in excess of the time of flight), a regime in which the diffusion approximation breaks down. Our image reconstruction method is based on extension of x-ray computed tomography (CT) to the optical regime. The CT algorithm must be generalized to take into account the distributions of photon paths. We express the point spread function (PSF) in terms of the Green's function for the transport equation. This PSF then provides weighting functions for use in a generalized series expansion method of x-ray CT. Experiments were performed on a turbid medium with scattering and absorption properties similar to those of human breast tissue. Multiple absorbers were embedded into the medium to mimic tumors. Coaxial transmission scans were collected in two projections, and the early-time portions were analyzed. Through accurate modeling, we could remove the blurring associated with multiple scattering and obtain high-resolution images. Our results show that the diffusion approximation PSF is inadequate to describe the early arriving photons. A PSF incorporating causality is required to reconstruct accurate images of turbid media.

Quantification of (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene adducts in human serum albumin by laser-induced fluorescence: implications for the in vivo metabolism of benzo[a]pyrene.

The ubiquitous environmental carcinogen benzo[a]pyrene (BaP) is metabolized in vivo in humans to its ultimate carcinogenic form of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Mouse skin tumorigenicity studies indicate that the (7R,8S,9S,10R) enantiomer of BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7R,8S,9S,10R)-BPDE], is a potent tumor initiator, whereas the (7S,8R,9R,10S) enantiomer of BPDE, (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7S,8R,9R,10S)-BPDE], may act as a tumor promoter. In vitro experiments have shown that human liver microsomes are capable of metabolizing BaP to both the (7R,8S,9S,10R) and (7S,8R,9R,10S) enantiomers of BPDE. However, the metabolism of BaP to (7S,8R,9R,10S)-BPDE has not been demonstrated in humans in vivo. The adducts formed between human serum albumin (HSA) and the (7S,8R,9R,10R) and (7R,8S,9S,10R) enantiomers of BPDE have been described previously. (7S,8R,9R,10S)-BPDE forms a stable adduct at histidine146 of HSA, whereas (7R,8S,9R,10R)-BPDE forms a relatively unstable ester adduct at aspartate187 or glutamate188 of HSA. Using high-performance liquid chromatography with laser-induced fluorescence (LIF) detector, we quantified the level of (7S,8R,9R,10S)-BPDE adducts at histidine146 in HSA isolated from 63 healthy males who were population control subjects for an ongoing case-control study of bladder cancer. By design, roughly half of the participants were lifelong nonsmokers (n = 35), whereas the remaining 28 participants were current smokers of varying intensities. HP-BPDE adducts were detected in 60 of the 63 samples (95%) by HPLC-LIF. Adduct levels ranged from undetectable (<0.04 fmol/mg HSA) to 0.77 fmol/mg HSA. The samples had a mean and median (7S,8R,9R,10S)-BPDE-HSA adduct level of 0.22 and 0.16 fmol of adduct/mg albumin, respectively. Mean adduct levels did not differ between smokers and nonsmokers (P = 0.72). Occupational exposure to polycyclic aromatic hydrocarbons was unrelated to adduct level (P = 0.62). Intake frequencies of two food items showed statistically significant associations with adduct levels. Consumption of sweet potatoes was negatively related to adduct level (P = 0.029), whereas intake of grapefruit juice was positively related to adduct level (P = 0.045). None of the three indices of residential ambient air pollution under study showed a statistically significant association with adduct levels.

Prospects for in vivo Raman spectroscopy.

Raman spectroscopy is a potentially important clinical tool for real-time diagnosis of disease and in situ evaluation of living tissue. The purpose of this article is to review the biological and physical basis of Raman spectroscopy of tissue, to assess the current status of the field and to explore future directions. The principles of Raman spectroscopy and the molecular level information it provides are explained. An overview of the evolution of Raman spectroscopic techniques in biology and medicine, from early investigations using visible laser excitation to present-day technology based on near-infrared laser excitation and charge-coupled device array detection, is presented. State-of-the-art Raman spectrometer systems for research laboratory and clinical settings are described. Modern methods of multivariate spectral analysis for extracting diagnostic, chemical and morphological information are reviewed. Several in-depth applications are presented to illustrate the methods of collecting, processing and analysing data, as well as the range of medical applications under study. Finally, the issues to be addressed in implementing Raman spectroscopy in various clinical applications, as well as some long-term directions for future study, are discussed.

Interferometric phase-dispersion microscopy.

We describe a new scanning microscopy technique, phase-dispersion microscopy (PDM). The technique is based on measuring the phase difference between the fundamental and the second-harmonic light in a novel interferometer. PDM is highly sensitive to subtle refractive-index differences that are due to dispersion (differential optical path sensitivity, 5 nm). We apply PDM to measure minute amounts of DNA in solution and to study biological tissue sections. We demonstrate that PDM performs better than conventional phase-contrast microscopy in imaging dispersive and weakly scattering samples.

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.

Near-infrared fluorescence spectroscopy detects Alzheimer's disease in vitro.

The purpose of this study was to investigate whether near-infrared (NIR) fluorescence spectroscopy could be used to detect Alzheimer's disease (AD) by brain tissue autofluorescence. Unfixed temporal cortex specimens from AD cases and age-matched, non-AD controls were frozen at autopsy and then thawed just prior to spectral measurement. Spectra of intrinsic tissue fluorescence induced by 647 nm light were recorded from 650 to 850 nm. We used principal component analysis of the tissue spectra from 17 AD cases and 5 non-AD control cases in a calibration study to establish a diagnostic algorithm. Retrospectively applied to the calibration set, the algorithm correctly classified 23 of 24 specimens. In a prospective study of 19 specimens from 5 AD brains and 2 non-AD control brains, 3 of the 4 control specimens and all AD specimens were correctly diagnosed. Both the excitation light used and the measured brain tissue autofluorescence are at NIR wavelengths that can propagate through skull and overlying tissue. Therefore, our results demonstrate an optical spectroscopic technique that carries direct molecular level information about disease. This is the first step toward a clinical tool that has the potential to be applied to the noninvasive diagnosis of AD in living patients.

Spatial coherence of forward-scattered light in a turbid medium.

We study spatially coherent forward-scattered light propagating in a turbid medium of moderate optical depth (0-9 mean free paths). Coherent detection was achieved by using a tilted heterodyne geometry, which desensitizes coherent detection of the attenuated incident light. We show that the degree of spatial coherence is significantly higher for light scattered only once in comparison with that for multiply scattered light and that it approaches a small constant value for large numbers of scattering events.