Following the Inter-Institute workshop on Optical Imaging from Bench to Bedside held at the National Institutes of Health in September 2000, Bruce Tromberg suggested that we publish a special section in JBO devoted to this topic, which would be sponsored by NIH.

Current dental diagnostic methods can detect caries but cannot quantify the mineral status of a lesion. Quantitative lightinduced fluorescence (QLF) measures the percentage of fluorescence change of demineralized enamel with respect to surrounding sound enamel, and relates it directly to the amount of mineral lost during demineralization. Development of caries-like lesions and subsequent remineralization of the lesions were monitored by QLF. The results showed that the percentage of fluorescence change (?Q) increased linearly with the demineralization time and decreased with increased remineralization time. Stained teeth were whitened with a bleaching agent and the change in stain intensity (?E ) was quantified using QLF. The results showed that ?E decreased linearly as the tooth regained its natural color. Factors that might affect the use of QLF to detect and quantify caries were also examined. It was concluded that QLF could be used to detect and longitudinally monitor the progression or remineralization of incipient caries, however lesion detection may be limited by the presence of saliva or plaque and enhanced by staining. The change in shade of discolored teeth by whitening agents could be quantitatively measured by QLF.

Fourteen BALB/c mice were divided into two groups. One group served as the control and the second group was injected with a squamous cell carcinoma cell line to the tongue. After tumor development (1–4 weeks), mice were injected with a FITC conjugated CD3 marker to their tongues. Immediately after the marker injection, the clearance of the marker was measured using a laser spectroscopy system. The markers were excited by an argon laser at 488 nm and the fluorescence signal was measured as a function of time. A biopsy was taken from every mouse after the procedure and the excised tissue was histologically evaluated. Analysis of clearance times revealed a second order exponential decay for both groups with a slower pace of signal clearance for the sick mice.

The objective of this study was to compare the effects of chemopreventive agents on natural fluorescence emission of ovarian cells in a cell culture and in a primate model as a feasibility trial to monitor drug activity. Methods: Fluorescence emission spectra were collected from normal (NOE) and immortalized ovarian surface epithelial cells at 290, 360, and 450 nm excitation. Redox potentials were calculated and compared to % apoptosis and cell survival. Fluorescence emission spectra were collected from 18 female rhesus macaques receiving fenretinide [N-(-hydroxyphenyl)retinamide (4- HPR)] orally and/or oral contraceptive pills (OCP) or no medication. Fluorescence intensities and redox ratios were compared using a twotailed Student’s t test. Results: Apoptosis and cell survival correlated with fluorescence emission consistent with metabolically active proteins [flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD(P)H)] and the resulting redox ratio in cells grown with 4-HPR. The 4-HPR consistently inhibited cell survival in a dose dependent manner. Degree of correlation varied between different cell lines. In primates receiving 4-HPR, fluorescence emission was increased at 450 nm excitation, 550 nm emission consistent with FAD presence, whereas those receiving OCP showed decreased emission at 350 nm excitation, 450 nm emission consistent with decreased NAD(P)H presence. Redox ratios were increased by both drugs. Conclusions: Fluorescence intensity and redox ratio appear to be altered by 4-HPR treatment in vivo and in cell culture and by OCP in vivo. Fluorescence intensity may be useful to monitor chemopreventive agents in clinical trials.

A novel, minimally invasive measurement technique has been developed for the detection of drugs in the anterior chamber of the eye. Presently there is no satisfactory, real-time detection method available to the ophthalmic community. Accurate determination of drug concentrations in the eye would be of great value and assistance to researchers and manufacturers of ophthalmic drugs and ocular implants, to enable a better understanding of intraocular pharmacokinetics. At present researchers use techniques of direct sampling of the aqueous humor from laboratory animal eyes into which the drug has been introduced topically or systemically. Rabbit eyes are frequently used in this context. Sampling via paracentesis is invasive, and does not yield a continuous measurement. Our approach to addressing this measurement requirement is, in effect, to turn the eye into a cuvette and use optical absorbance spectroscopy measurements to detect drug concentrations. A novel contact lens has been designed using commercial, off-the-shelf, optical design software. The lenses have been optimized to direct light across the anterior chamber of a rabbit’s eye. Practical demonstration and characterization of light propagation across the eye have been undertaken and are reported. Preliminary results of the identification of drug compounds introduced into model eyes are also reported.

Hemorrhagic hypovolemia and inotropic agent administration were used to manipulate cardiac output (CO) and oxygen delivery in rabbits to investigate the correlation between noninvasive frequency domain photon migration (FDPM) spectroscopy and invasive hemodynamic monitoring parameters. Frequency-domain photon migration provides quantitative measurements of light absorption and reduced scattering (µa and µ's, respectively) in tissue. Wavelength dependent µa values were used to calculate in vivo tissue concentration of deoxyhemoglobin [Hb], oxyhemoglobin [HbO2], total hemoglobin [TotHb], and water [H2O] as well as mixed arterial-venous oxygen saturation (StO2) in tissue. FDPM-derived physiologic properties were correlated with invasive measurements of CO and mean pulmonary artery pressure (mPAP), FDPM-derived [TotHb] and StO2 correlated significantly with hemorrhaged volume (HV), mPAP, and CO. Correlation coefficients for [TotHb] vs HV, mPAP, and CO were ?0.77, 0.86, and 0.70, respectively. Correlation coefficients of StO2 vs HV, mPAP, and CO were ?0.71, 0.55, and 0.61, respectively. Dobutamine induced changes resulted in correlation coefficients between FDPM-derived and invasively measured physiologic parameters that are comparable to those induced by hypovolemia. FDPM spectroscopy is sensitive to changes in mPAP and CO of as little as 15%. These results suggest that FDPM spectroscopy may be used in clinical settings to noninvasively monitor central hemodynamic parameters and to directly assess oxygenation of tissues.

Wavelength selection and prediction algorithm for determining total hemoglobin concentration are investigated. A model based on the difference in optical density induced by the pulsation of the heart beat is developed by taking an approximation of Twersky’s theory on the assumption that the variation of blood vessel size is small during arterial pulsing. A device is constructed with a fivewavelength light emitting diode array as the light source. The selected wavelengths are two isobestic points and three in compensation for tissue scattering. Data are collected from 129 outpatients who are randomly grouped as calibration and prediction sets. The ratio of the variations of optical density between systole and diastole at two different wavelengths is used as a variable. We selected several such variables that show high reproducibility among all variables. Multiple linear regression analysis is made in order to predict total hemoglobin concentration. The correlation coefficient is 0.804 and the standard deviation is 0.864 g/dL for the calibration set. The relative percent error and standard deviation of the prediction set are 8.5% and 1.142 g/dL, respectively. We successfully demonstrate the possibility of noninvasive hemoglobin measurement, particularly, using the wavelengths below 1000 nm.

Using the modified Lambert-Beer law to analyze attenuation changes measured noninvasively during functional activation of the brain might result in an insufficient separation of chromophore changes (‘‘cross talk’’) due to the wavelength dependence of the partial pathlength of photons in the activated volume of the head. The partial pathlength was estimated by performing Monte Carlo simulations on layered head models. When assuming cortical activation (e.g., in the depth of 8–12 mm), we determine negligible cross talk when considering changes in oxygenated and deoxygenated hemoglobin. But additionally taking changes in the redox state of cytochrome-c-oxidase into account, this analysis results in significant artifacts. An analysis developed for changes in mean time of flight— instead of changes in attenuation—reduces the cross talk for the layers of cortical activation. These results were validated for different oxygen saturations, wavelength combinations and scattering coefficients. For the analysis of changes in oxygenated and deoxygenated hemoglobin only, low cross talk was also found when the activated volume was assumed to be a 4-mm-diam sphere.

Near-infrared (NIR) diffuse optical spectroscopy and imaging may enhance existing technologies for breast cancer screening, diagnosis, and treatment. NIR techniques are based on quantitative measurements of functional contrast between healthy and diseased tissue. In this study we measured the spectral dependence of tissue absorption (µa ) and reduced scattering (µ's) in the breasts of 30 healthy women and one woman with a fibroadenoma using a sevenwavelength frequency-domain photon migration probe. Subjects included pre- and postmenopausal women between the ages of 18 and 64. Multi-spectral measurements were used along with a fourcomponent fit to determine the concentrations of de-oxy and oxyhemoglobin, water and lipids in breast. The scattering spectral shape was also quantified. Our measurements demonstrate that the measured concentrations of NIR analytes correlate well with known breast physiology. Although the tissue scattering at a single wavelength was found to have little value as a functional parameter, the dependence of the scattering on wavelength provided key insights into breast composition and physiology. Lipids and scattering spectra in the breast were found to increase and decrease, respectively, with increasing body mass index. Simple calculations are also provided to demonstrate potential penalties from ignoring the contributions of water and lipids in breast measurements. Finally, water is shown to be a possible indicator for detecting a fibroadenoma, whereas the hemoglobin saturation was found to be a poor indicator. Multi-spectral measurements, compared to measurements restricted to one or two wavelengths, provide additional information that may be useful in managing breast disease.

Images of hemoglobin concentration and oxygen saturation are presented from multispectral near-infrared tomographic measurements in the breast of a woman with an invasive cancer. Images of the absorption coefficient and reduced scattering coefficient are recovered from the measured data using a finite element reconstruction algorithm based on the frequency-domain diffusion equation. Three methods of recovering the hemoglobin concentration and oxygen saturation images are presented which compensate for water and lipid absorption in different ways: (1) an assumed bulk content of water and lipids is used, (2) four chromophores are imaged, and (3) scattering power data are applied to deduce water and lipid images. In all three cases, a large increase in the hemoglobin concentration (3:1) is observed at the location of the cancer while a maximum of 15% difference is observed in the hemoglobin images between each of these methods for water and lipid compensation.

For the first time we use a random walk methodology based on time-dependent contrast functions to quantify the optical properties of breast tumors (invasive ductal carcinoma) of two patients. Previously this theoretical approach was successfully applied for analysis of embedded objects in several phantoms. Data analysis was performed on distributions of times of flight for photons transmitted through the breast which were recorded in vivo using a time-domain scanning mammograph at 670 and 785 nm. The size of the tumors, their optical properties, and those of the surrounding tissue were reconstructed at both wavelengths. The tumors showed increased absorption and scattering. From the absorption coefficients at both wavelengths blood oxygen saturation was estimated for the tumors and the surrounding tissue.

We present for the first time full three-dimensional (3D) volumetric reconstruction of absorption images of in vitro and in vivo bones and joints from near-infrared tomographic measurements. Imaging experiments were conducted on human finger and chicken bones embedded in cylindrical scattering media using a Clemson multichannel diffuse optical imager. The volumetric optical images were recovered with our 3D finite element based reconstruction algorithm. Our results show that 3D imaging methods can provide details of the joint structure/composition that would be impossible from twodimensional imaging methods.

We demonstrate that the effects of cell–cell contact and of changes in cell shape have only a minor effect on the angular distribution of light scattering from mammalian fibroblast cells. This result is important for the development of light scattering as a noninvasive tool for tissue diagnostics such as cancer detection. Changes in cell organization that are not accompanied by changes in internal cellular structure may not be measurable. On the other hand, changes in internal cellular structure should be measurable without interference from changes in overall cellular organization. The second major result of this work is that there are small but significant differences between light scattering of tumorigenic and nontumorigenic cells grown in a three-dimensional culture system. The cause of the differences in light scattering are likely due to the nontumorigenic cells arresting in the G1 phase of the cell cycle, while the tumorigenic cells continue to proliferate.

Several investigations were performed in order to develop an in vivo endoscopic method to differentiate between malignant and healthy tissue working on the assumption that each diseased state of biological tissue has its own characteristic infrared (IR) spectral pattern. The technical design of the laboratory setup is presented here together with the experimental details and the results. Two regions (1245–1195) and (1045–995) cm?1 within the fingerprint (<1500 cm?1) region were selected for analysis. Lead salt diode lasers were used as excitation sources and IR radiation was transmitted via silver halide wave guides to the tissue to be investigated. The IR radiation is returned to a mercury–cadmium–telluride detector by another IR cable. The measurements were carried out in attenuated total reflectance and diffuse reflection/remission. Human colon carcinoma tissue, under humid conditions, was used as a target for experiments to simulate in vivo conditions. Specimens were mapped using a stepper, motor powered, x /y /z -translation stage with a spatial resolution of 1 mm. The results were compared with similar measurements from a Fourier transform IR (FTIR) interferometer/FTIR microscope setup in the wave number region of 4000–900 cm?1.

Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique in which a fluorescent labeled probe hybridizes to a target nucleotide sequence of deoxyribose nucleic acid. Upon excitation, each chromosome containing the target sequence produces a fluorescent signal (spot). Because fluorescent spot counting is tedious and often subjective, automated digital algorithms to count spots are desirable. New technology provides a stack of images on multiple focal planes throughout a tissue sample. Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in single-focalplane methods. This paper proposes an algorithm for global spot counting in stacked three-dimensional slice FISH images without the necessity of nuclei segmentation. It is designed to work in complex backgrounds, when there are agglomerated nuclei, and in the presence of illumination gradients. It is based on the morphological tophat transform, which locates intensity spikes on irregular backgrounds. After finding signals in the slice images, the algorithm groups these together to form three-dimensional spots. Filters are employed to separate legitimate spots from fluorescent noise. The algorithm is set in a comprehensive toolbox that provides visualization and analytic facilities. It includes simulation software that allows examination of algorithm performance for various image and algorithm parameter settings, including signal size, signal density, and the number of slices.

Color Doppler optical coherence tomography (CDOCT) is a functional extension of optical coherence tomography (OCT) that can image flow in turbid media. We have developed a CDOCT system capable of imaging flow in real time. Doppler processing of the analog signal is accomplished in hardware in the time domain using a novel autocorrelation technique. This Doppler processing method is compatible with a high speed OCT system capable of imaging in real time. Using this system, we demonstrate cross-sectional imaging of bidirectional flow with CDOCT at four frames per second in a tissuesimulating phantom consisting of intralipid solution flowing in glass capillaries. As a demonstration of real-time imaging of blood flow in vivo we imaged pulsatible blood flow in a rat femoral artery at eight frames per second. Issues of velocity sensitivity, imaging speed, and range of velocity measurement are discussed, as well as potential applications of real-time CDOCT.

The closed-form solution of the so-called statistical multivariate calibration model is given in terms of the pure component spectral signal, the spectral noise, and the signal and noise of the reference method. The ‘‘statistical’’ calibration model is shown to be as much grounded on the physics of the pure component spectra as any of the ‘‘physical’’ models. There are no fundamental differences between the two approaches since both are merely different attempts to realize the same basic idea, viz., the spectrometric Wiener filter. The concept of the application-specific signal-to-noise ratio (SNR) is introduced, which is a combination of the two SNRs from the reference and the spectral data. Both are defined and the central importance of the latter for the assessment and development of spectroscopic instruments and methods is explained. Other statistics like the correlation coefficient, prediction error, slope deficiency, etc., are functions of the SNR. Spurious correlations and other practically important issues are discussed in quantitative terms. Most important, it is shown how to use a priori information about the pure component spectra and the spectral noise in an optimal way, thereby making the distinction between statistical and physical calibrations obsolete and combining the best of both worlds. Companies and research groups can use this article to realize significant savings in cost and time for development efforts.

We introduce new realistic three-dimensional tissue phantoms which can help to understand the optical properties of human epithelium as well as the optical signatures associated with the dysplasia to carcinoma sequence. The phantoms are based on a step by step multilayer reconstitution of the epithelial tissue using main components characteristic for the human epithelium. Each consecutive step is aimed to increase the similarity between real tissue and a phantom. We began by modeling the stromal layer which predominantly consists of a network of collagen bundles. Phantoms consisting of a collagen matrix alone and in the presence of embedded cervical cells were created. Their morphology and fluorescence properties were studied and were compared with those of cervical epithelium. We show that the phantoms resemble the microstructure and the optical properties of the human epithelial tissue.We also demonstrate that the proposed phantoms provide an opportunity to study changes in optical properties of different tissue components as a result of their interactions with each other or exogenous factors.