Frequency-domain optical mammography has been advocated to improve contrast and thus cancer detectability in breast transillumination. To the best of our knowledge, this report provides the first systematic clinical results of a frequency-domain laser scanning mammograph (FLM). The instrument provides monochromatic light at 690 and 810 nm, whose intensity is modulated at 110.0010 and 110.0008 MHz, respectively. The breast is scanned by stepwise positioning of source and detector, and amplitude and phase for both wavelengths are measured by a photomultiplier tube using heterodyne detection. Images are formed representing amplitude or phase data on linear gray scales. Furthermore, various algorithms carrying on more than one signal (amplitude ratio, phase difference, µa , µ's , N) were essayed. Twenty visible cancers out of 25 cancers in the first 59 investigations were analyzed for their quantitative contrast with respect to the whole breast or to defined reference areas. Contrast definitions refer to the signal itself (definition 1), to the signal noise (definition 2), or were based on nonparametric comparison (definition 3). The amplitude signal provides better contrast than the phase signal. Ratio images between red and infrared amplitudes gave variable results; in some cases the tumor contrast was canceled. The algorithms to determine ma and ms8 from amplitude and phase data did not significantly improve upon objective contrast. The N algorithm, using the phase signal to flatten the amplitude signal did significantly improve upon contrast according to contrast definitions 1 and 2, however, did not improve upon nonparametric contrast. Thus, with the current instrumentation, the phase signal is helpful to correct for the complex and variable geometry of the breast. However, an independent informational content for tumor differentiation could not be determined. The flat field algorithm did greatly enhance optical contrast in comparison with amplitude or amplitude ratio images. Further evaluation of FLM will have to be based on the N-algorithm images.

By applying linear perturbation theory to the radiation transport equation, the inverse problem of optical diffusion tomography can be reduced to a set of linear equations, Wµ=R, where W is the weight function, µ are the cross-section perturbations to be imaged, and R is the detector readings perturbations. We have studied the dependence of image quality on added systematic error and/or random noise in W and R. Tomographic data were collected from cylindrical phantoms, with and without added inclusions, using Monte Carlo methods. Image reconstruction was accomplished using a constrained conjugate gradient descent method. Results show that accurate images containing few artifacts are obtained when W is derived from a reference state whose optical thickness matches that of the unknown test medium. Comparable image quality was also obtained for unmatched W, but the location of the target becomes more inaccurate as the mismatch increases. Results of the noise study show that image quality is much more sensitive to noise in W than in R, and the impact of noise increases with the number of iterations. Images reconstructed after pure noise was substituted for R consistently contain large peaks clustered about the cylinder axis, which was an initially unexpected structure. In other words, random input produces a nonrandom output. This finding suggests that algorithms sensitive to the evolution of this feature could be developed to suppress noise effects.

Tissue spectroscopy and endoscopy are combined with a tissue site-selective fluorescent probe molecule to demonstrate in vitro, spatial, remote, quantitative imaging of the rat small intestine. The probe molecule employed, Tb-3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca- 1(15),11,13-triene (Tb–PCTMB), is shown to bind with the small intestine and provide improved image contrast. High sensitivity is possible due to the absorption-emission Stokes’s shift exhibited by the Tb–PTCMB complex. Excitation is centered near 270 nm and multifeatured emission is observed at 490, 550, 590, and 625 nm. Sprague-Dawley rats were dosed with the Tb-PTCMB complex, which shows biodistribution, leading to preferential binding to the inner surface of the small intestine. It is shown that the fluorescent image, taken at 550 nm, can be used to quantify the amount of Tb–PCTMB present in an excised tissue sample. The 3s detection limits are found to be in the femtomole range. An optical mass balance for Tb–PCTMB-dosed small intestine is performed and along with radiotracer biodistribution, demonstrates that approximately 40% of the marker probe resides in the endothelial tissue of the small intestine inner lumen. This result is of particular interest since most adult colon cancers develop in this region. These results demonstrate the ability to perform spatial, quantitative, in vitro, endoscopic imaging of a complex biological sample using a probe marker.

The combined use of fluorescence resonance energy transfer (FRET) microscopy and expression of genetic vectors encoding protein fusions with green fluorescent protein (GFP) and blue fluorescent protein (BFP) provides an exceptionally sensitive method for detecting the interaction of protein partners in living cells. The acquisition of FRET signals from GFP- and BFP-fusion proteins expressed in living cells was demonstrated using an optimized imaging system and high sensitivity charge coupled device camera. This imaging system was used to detect energy transfer signals from a fusion protein containing GFP physically linked to BFP expressed in living HeLa cells. In contrast, the co-localization of noninteracting GFP- and BFP-fusion proteins was not sufficient for energy transfer. The FRET imaging system was then used to demonstrate dimerization of the pituitary-specific transcription factor Pit-1 within the living cell nucleus.

The possibility of mathematically describing the body surface represents a useful tool for several medical sectors, such as prosthetics or plastic surgery, and could improve diagnosis and objective evaluation of deformities and the follow-up of progressive diseases. The approach presented is based on the acquisition of a surface scanned by a laser beam. The 3-D coordinates of the spot generated on the surface by the laser beam are computed by an automatic image analyzer (ELITE system). Using at least two different views of the subject, the 3-D coordinates are obtained by stereophotogrammetry. A software package for graphic representation and extraction of linear superficial and volumetric features from the acquired surface has been developed and some preliminary results with mammary reconstruction are presented. A good mammary reconstruction after mastectomy must achieve two results. First, the reconstruction should follow the patients’ wishes and second, the reconstructed breast should be as similar as possible to the contralateral one (symmetry is the most important aesthetic parameter to be considered). To achieve these goals, a knowledge of breast volume, area, and shape features are essential for the surgeon. In such a context, this system could be a valuable tool in improving breast reconstructive surgery.

Fluorescence spectra measured from biological samples, such as tissues or cell suspensions, are usually distorted due to the light absorption by intrinsic chromophores. These distortions are aggravated by strong scattering of light inside the samples. A new method is described for a fast correction of these spectral distortions, using only steady-state spectroscopic measurements. The method is based on the formulas derived from a simplified photon diffusion model, in the isotropic one-dimensional approximation applied to a semi-infinite, highly scattering, and moderately absorbing medium with a refractive-index-matched boundary. The formulas describe the spectral distortions of the fluorescence emission and excitation spectra, together with the diffuse reflectance spectrum, as the functions of one spectral characteristic of the medium, the darkness, which is the ratio of absorption coefficient and reduced scattering coefficient. The algorithm does not involve any iterative procedures, and offers a direct, simple, and fast method for real-time spectral correction. The true fluorescence emission or excitation spectrum is directly calculated from a pair of experimental spectra: the fluorescence emission or excitation spectrum and the diffuse reflectance spectrum, measured from the same position on a sample. The correction produces the profile of the true fluorescence spectrum, the same as the one measured from the corresponding sample with an infinitely low absorption and no scattering. The restoration of the spectral profiles of true fluorescence emission and excitation spectra was tested experimentally, using highly scattering phantoms with a fluorescent dye and a deliberately added nonfluorescent dye producing strong inner-filter distortions.

A detectable signal is obtained from a laser Doppler flowmeter operating in the heterodyne mode with nano-and pico-second pulse laser sources. The ultrashort pulse probing may be useful for depth-dependent time-resolved laser Doppler velocity measurements of blood perfusion in biological tissues.

The mechanisms of myocardial oxygenation during reactive hyperemia were studied in the beating heart using continuous near infrared (NIR) spectroscopy. In open chest dogs, NIR spectroscopy was used to monitor brief occlusions of the left anterior descending artery. These occlusions produced a precipitous drop in tissue oxygen stores (tHbO2+MbO2), tissue blood volume, and the oxidation level of mitochondrial cytochrome a,a3 . Reperfusion produced a rapid increase in the NIR signals to supranormal levels, followed by gradual return to baseline. When the duration of occlusion was increased from 20 to 120 s, an essentially linear increase was produced in the overshoot areas defined by the NIR signals. Near infrared spectroscopy (NIRS) separated reactive hyperemia into two phases according to the tissue level of deoxyhemoglobin and deoxymyoglobin (tHb+Mb): (1) an early phase during which the tHb+Mb level was supranormal, reflecting enhanced O2 extraction; and (2) a late phase during which the tHb+Mb level was below baseline, reflecting decreased O2 extraction and increased tissue O2 availability. During reactive hyperemia, when O2 availability was maximal by NIR spectroscopy, O2 consumption was elevated but submaximal, indicating that MVO2 was not limited by O2 availability. Cytochrome a,a3 oxidation state also was restored fully. Thus, myocardial oxygenation is highly regulated during reactive hyperemia. Cellular O2 supply and mitochondrial oxidation state are restored early during reactive hyperemia by increased O2 delivery, increases in tissue blood volume and enhanced O2 extraction.

The results of the experimental investigation of autofluorescence spectra of human skin in vivo caused by the UV radiation of the skin and by the external mechanical pressure applied to the skin are presented. These results are compared with results of Monte Carlo modeling of the autofluorescence of the skin with variable blood content. The proposed simple model of the skin gives the possibility of evaluation of changes of the blood and melanin content within the skin.

We derive a multipole scattering solution for a system resembling a simple cell. In the model, a spherical cytoplasm is surrounded by a concentric cell membrane. Contained within the cytoplasm is a nonconcentric spherical nucleus. Because of the nature of the (multipole expansion) solution, numerical results can be acquired quite rapidly. We show that the resulting scatter is very sensitive to the system geometry and optical properties. Such a solution may also be used to calculate the scatter from fluorescing molecules located within the cell.