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Issue 6 | November 2004 | pp. 1120-1374

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November 2004

Volume 9, Issue 6, pp. 1120-1374

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Editorial

Select this Article		JBO to Begin “e-First” Publication in 2005 Bruce J. Tromberg J. Biomed. Opt. 9, 1120 (2004); http://dx.doi.org/10.1117/1.1819951 Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
		Abstract Unavailable

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Special Section on Optics in Breast Cancer

Select this Article OPEN ACCESS		Guest Editorial: Special Section on Optics in Breast Cancer Sergio Fantini, Ph.D., K. Thomas Moesta, M.D., and Brian W. Pogue, Ph.D. J. Biomed. Opt. 9, 1121 (2004); http://dx.doi.org/10.1117/1.1813446 \| Cited 2 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract This Special Section Guest Editorial provides an overview of the topical area and an introduction to the articles featured in the special section.

Select this Article		Elastic scattering spectroscopy for intraoperative determination of sentinel lymph node status in the breast Kristie S. Johnson, Dennis W. Chicken, David C. O. Pickard, Andrew C. Lee, Gavin Briggs, Mary Falzon, Irving J. Bigio, Mohammed R. Keshtgar, and Stephen G. Bown J. Biomed. Opt. 9, 1122 (2004); http://dx.doi.org/10.1117/1.1802191 \| Cited 15 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract The ability to provide the best treatment for breast cancer depends on establishing whether or not the cancer has spread to the lymph nodes under the arm. Conventional assessment requires tissue removal, preparation, and expert microscopic interpretation. In this study, elastic scattering spectroscopy (ESS) is used to interrogate excised nodes with pulsed broadband illumination and collection of the backscattered light. Multiple spectra are taken from 139 excised nodes (53 containing cancer) in 68 patients, and spectral analysis is performed using a combination of principal component analysis and linear discriminant analysis to correlate the spectra with conventional histology. The data are divided into training and test sets. In test sets containing spectra from only normal nodes and nodes with complete replacement by cancer, ESS detects the spectra from cancerous nodes with 84% sensitivity and 91% specificity (per-spectrum analysis). In test sets that included normal nodes and nodes with partial as well as complete replacement by cancer, ESS detects the nodes with cancer with an average sensitivity of 75% and specificity of 89% (per-node analysis). These results are comparable to those from conventional touch imprint cytology and frozen section histology, but do not require an expert pathologist for interpretation. With automation of the technique, results could be made available almost instantaneously. ESS is a promising technique for the rapid, accurate, and straightforward detection of metastases in excised sentinel lymph nodes. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Intraoperatively assessed optical properties of malignant and healthy breast tissue used to determine the optimum wavelength of contrast for optical mammography R. L. P. van Veen, H. J. C. M. Sterenborg, A. W. K. S. Marinelli, and M. Menke-Pluymers J. Biomed. Opt. 9, 1129 (2004); http://dx.doi.org/10.1117/1.1803547 \| Cited 16 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We use spatially resolved diffuse remittance spectroscopy (DRS) for the measurement of absorption (μ_a) and reduced scattering (μs′) coefficients of normal and malignant breast tissue in vivo during surgery. Prior to these measurements, the linearity of the measurement technique was evaluated on liquid optical phantoms. In addition, the reproducibility of in-vivo tissue measurements was determined on a healthy volunteer. We present results of the in-vivo measurement of optical properties in the wavelength range from 600 to 1100 nm performed during radical mastectomy. A total of 24 patients were included in the study. Both the absorption and reduced scattering properties show large variations. Significant differences in optical properties between normal (glandular plus lipid rich tissue) and tumor tissues are present in 74% of all patients. However, in some cases the tumor showed lower values than normal tissue, and in other cases this was the other way around. Thus, a general trend in optical properties is not observed. However, the average absorption contrast of all patients as a function of wavelength reveals an optimal contrast peak at 650 nm. We believe that this relates to a difference in vascular saturation between tumor and adjacent normal tissue. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Bulk optical properties and tissue components in the female breast from multiwavelength time-resolved optical mammography Lorenzo Spinelli, Alessandro Torricelli, Antonio Pifferi, Paola Taroni, Gian Maria Danesini, and Rinaldo Cubeddu J. Biomed. Opt. 9, 1137 (2004); http://dx.doi.org/10.1117/1.1803546 \| Cited 44 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We present the results of a clinical study about optical properties and bulk composition of the female breast. The clinical study involved more than 150 subjects that underwent optical mammography. A multiwavelength time-resolved mammograph designed to collect time-resolved transmittance images of the breast at different wavelengths in the range 637 to 980 nm is used to this purpose. From the absorption spectrum of the breast, the concentrations of the main tissue constituents, i.e., oxygenated and deoxygenated hemoglobin, lipid, and water, are obtained for a subset of 113 breasts. The lipid content of breast is estimated for the first time on such a large number of subjects. The total hemoglobin concentration, blood oxygen saturation, lipid, and water content of breast is correlated to demographic information collected during the trial. As expected, breast optical properties and components undergo huge variations among different subjects. Different constituents, however, show interesting correlation with clinical parameters such as age, breast size, body mass index, and mammographic parenchymal pattern. These results suggest that optical measurements on breasts can be exploited to obtain relevant information on breast tissue composition. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Spectroscopic time-resolved diffuse reflectance and transmittance measurements of the female breast at different interfiber distances Antonio Pifferi, Johannes Swartling, Ekaterine Chikoidze, Alessandro Torricelli, Paola Taroni, Andrea Bassi, Stefan Andersson-Engels, and Rinaldo Cubeddu J. Biomed. Opt. 9, 1143 (2004); http://dx.doi.org/10.1117/1.1802171 \| Cited 29 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract The first, to our knowledge, in-vivo broadband spectroscopic characterization of breast tissue using different interfiber distances as well as transmittance measurements is presented. Absorption and scattering properties are measured on six healthy subjects, using time-resolved diffuse spectroscopy and an inverse model based on the diffusion equation. Wavelength-tunable picosecond-pulse lasers and time-correlated single-photon counting detection are employed, enabling fully spectroscopic measurements in the range 610 to 1040 nm. Characterization of the absorption and reduced scattering coefficients of breast tissue is made with the aim of investigating individual variations, as well as variations due to different measurement geometries. Diffuse reflectance measurements at different interfiber distances (2, 3, and 4 cm) are performed, as well as measurements in transmittance mode, meaning that different sampling volumes are involved. The results show a large variation in the absorption and scattering properties depending on the subject, correlating mainly with the water versus lipid content of the breast. Intrasubject variations, due to different interfiber distances or transmittance modes, correlate with the known structures of the breast, but these variations are small compared to the subject-to-subject variation. The intrasubject variations are larger for the scattering data than the absorption data; this is consistent with different spatial localization of the measurements of these parameters, which is explained by the photon migration theory. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Near-infrared imaging of the human breast: complementing hemoglobin concentration maps with oxygenation images Erica Heffer, Vivian Pera, Oliver Schütz, Horst Siebold, and Sergio Fantini J. Biomed. Opt. 9, 1152 (2004); http://dx.doi.org/10.1117/1.1805552 \| Cited 25 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We have previously reported a comparison between edge-corrected near-infrared optical mammograms and those that have undergone a further image-processing step based on a spatial second derivative. In this work, we go a step further by combining the second-derivative images from four wavelengths (690, 750, 788, and 856 nm) to obtain oxygenation-index images. While the spatial second derivative improves contrast and allows for visibility of fine structures in the images, thereby improving the sensitivity to tumor detection, additional information is needed to avoid false-positive results. The oxygenation-index images are introduced to address this issue. Oxygenation information may help discriminate benign from malignant breast lesions, thereby effectively complementing single-wavelength optical mammograms that display optically dense regions within the breast with high sensitivity. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Improved quantification of small objects in near-infrared diffuse optical tomography Subhadra Srinivasan, Brian W. Pogue, Hamid Dehghani, Shudong Jiang, Xiaomei Song, and Keith D. Paulsen J. Biomed. Opt. 9, 1161 (2004); http://dx.doi.org/10.1117/1.1803545 \| Cited 17 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Diffuse optical tomography allows quantification of hemoglobin, oxygen saturation, and water in tissue, and the fidelity in this quantification is dependent on the accuracy of optical properties determined during image reconstruction. In this study, a three-step algorithm is proposed and validated that uses the standard Newton minimization with Levenberg-Marquardt regularization as the first step. The second step is a modification to the existing algorithm using a two-parameter regularization to allow lower damping in a region of interest as compared to background. This second stage allows the recovery of the actual size of an inclusion. A region-based reconstruction is the final third step, which uses the estimated size and position information from step 2 to yield quantitatively accurate average values for the optical parameters. The algorithm is tested on simulated and experimental data and is found to be insensitive to object contrast and position. The percentage error between the true and the average recovered value for the absorption coefficient in test images is reduced from 47 to 27% for a 10-mm inclusion, from 38 to 13% for a 15-mm anomaly, and from 28 to 5.5% for a 20-mm heterogeneity. Simulated data with absorbing and scattering heterogeneities of 15 mm diam located in different positions show recovery with less than 15% error in absorption and 6% error in reduced scattering coefficients. The algorithm is successfully applied to clinical data from a subject with a breast abnormality to yield quantitatively increased absorption coefficients, which enhances the contrast to 3.8 compared to 1.23 previously. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Photoacoustic mammography laboratory prototype: imaging of breast tissue phantoms Srirang Manohar, Alexei Kharine, Johan C. G. van Hespen, Wiendelt Steenbergen, and Ton G. van Leeuwen J. Biomed. Opt. 9, 1172 (2004); http://dx.doi.org/10.1117/1.1803548 \| Cited 36 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We present a laboratory version of a photoacoustic mammoscope, based on a parallel plate geometry. The instrument is built around a flat high-density ultrasound detector matrix. The light source is a Q-switched Nd:YAG laser with a pulse duration of 5 ns. To test the instrument, a novel photoacoustic phantom is developed using poly(vinyl alcohol) gel, prepared by a simple procedure that imparts optical scattering suggestive of breast tissue to it without the requirement for extraneous scattering particles. Tumor simulating poly(vinyl alcohol) gel spheres appropriately dyed at the time of preparation are characterized for optical absorption coefficients. These are then embedded in the phantom to serve as tumors with absorption contrasts ranging from 2 to 7, with respect to the background. Photoacoustic studies in transmission mode are performed, by acquiring the laser-induced ultrasound signals from regions of interest in the phantom. Image reconstruction is based on a delay-and-sum beamforming algorithm. The results of these studies provide an insight into the capabilities of the prototype. Various recommendations that will guide the evolving of our laboratory prototype into a clinical version are also discussed. © 2004 Society of Photo-Optical Instrumentation Engineers.

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Research Papers

Select this Article		Measurement of bacterial concentration fractions in polymicrobial mixtures by Raman microspectroscopy Qingyuan Zhu, Robert G. Quivey, and Andrew J. Berger J. Biomed. Opt. 9, 1182 (2004); http://dx.doi.org/10.1117/1.1803844 \| Cited 8 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Relative concentrations of Streptococcus mutans and Streptococcus sanguis are important parameters in the study of dental caries, but current methods of measuring these concentrations are time consuming and prone to inaccuracies. We investigate the use of Raman spectroscopy for measuring relative concentrations of these two bacterial species in solid mixtures. To our knowledge, this is the first time Raman spectroscopy has been used to analyze bacterial mixtures rather than to identify the species of a pure colony. Mixtures of the two streptococcal species in various ratios are measured for 200 s using a home-built Raman microscope. Spectral correlations with bacterial content were identified via partial least-squares analysis. The relative concentrations of S. mutans in subsequent samples are predicted with a root mean squared error below 5%. In clinical plaque samples, this sort of accuracy would enable discrimination between normal and dangerously elevated levels of S. mutans. Samples with and without salivary proteins are predicted with equal accuracy. This result shows the potential of Raman spectroscopy for analyzing mixed populations of bacteria, such as those that occur in oral plaques. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Raman microspectroscopic mapping studies of human bronchial tissue Senada Koljenović, Tom C. Bakker Schut, Jan P. van Meerbeeck, Alexander P. W. M. Maat, Sjaak A. Burgers, Pieter E. Zondervan, Johan M. Kros, and Gerwin J. Puppels J. Biomed. Opt. 9, 1187 (2004); http://dx.doi.org/10.1117/1.1805555 \| Cited 25 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Characterization of the biochemical composition of normal bronchial tissue is a prerequisite for understanding the biochemical changes that accompany histological changes during lung cancer development. In this study, 12 Raman microspectroscopic mapping experiments are performed on frozen sections of normal bronchial tissue. Pseudocolor Raman images are constructed using principal component analysis and K-means cluster analysis. Subsequent comparison of Raman images with histologic evaluation of stained sections enables the identification of the morphologic origin (e.g., bronchial mucus, epithelium, fibrocollagenous stroma, smooth muscle, glandular tissue, and cartilage) of the spectral features. Raman spectra collected from the basal side of epithelium consistently show higher DNA contributions and lower lipid contributions when compared with superficial epithelium spectra. Spectra of bronchial mucus reveal a strong signal contribution of lipids, predominantly triolein. These spectra are almost identical to the spectra obtained from submucosal glands, which suggests that the bronchial mucus is mainly composed of gland secretions. Different parts of fibrocollagenous tissue are distinguished by differences in spectral contributions from collagen and actin/myosin. Cartilage is identified by spectral contributions of glycosaminoglycans and collagen. As demonstrated here, in situ analysis of the molecular composition of histologic structures by Raman microspectroscopic mapping creates powerful opportunities for increasing our fundamental understanding of tissue organization and function. Moreover, it provides a firm basis for further in vitro and in vivo investigations of the biochemical changes that accompany pathologic transformation of tissue. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Raman spectroscopy of in vivo cutaneous melanin Zhiwei Huang, Harvey Lui, X. K. Chen, Abdulmajeed Alajlan, David I. McLean, and Haishan Zeng J. Biomed. Opt. 9, 1198 (2004); http://dx.doi.org/10.1117/1.1805553 \| Cited 25 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We successfully acquire the in vivo Raman spectrum of melanin from human skin using a rapid near-infrared (NIR) Raman spectrometer. The Raman signals of in vivo cutaneous melanin are similar to those observed from natural and synthetic eumelanins. The melanin Raman spectrum is dominated by two intense and broad peaks at about 1580 and 1380 cm⁻¹, which can be interpreted as originating from the in-plane stretching of the aromatic rings and the linear stretching of the C-C bonds within the rings, along with some contributions from the C-H vibrations in the methyl and methylene groups. Variations in the peak frequencies and bandwidths of these two Raman signals due to differing biological environments have been observed in melanin from different sources. The ability to acquire these unique in vivo melanin signals suggests that Raman spectroscopy may be a useful clinical method for noninvasive in situ analysis and diagnosis of the skin. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Determination of the modulation transfer function for a time-gated fluorescence imaging system Sarah Gundy, Wil Van der Putten, Andy Shearer, Daniel Buckton, and Alan G. Ryder J. Biomed. Opt. 9, 1206 (2004); http://dx.doi.org/10.1117/1.1803550 Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract The use of fluorescence for cancer detection is currently under investigation. Presently, steady-state fluorescence detection methods are in use, but have limitations due to poor contrast between the fluorescence of the tumor and background autofluorescence. Improved contrast can be obtained with time-resolved techniques because of the differing lifetimes between autofluorescence and exogenous photosensitizers that selectively accumulate within tumor tissue. An imaging system is constructed using a fast-gated (200-ps) charge-coupled device (CCD) camera and a pulsed 635-nm laser diode. To characterize the ability of the system to transfer object contrast to an image, the modulation transfer function (MTF) of the system is acquired by employing an extended knife-edge technique. A knife-edge target is assembled by drilling a rectangular well into a block of polymethyl methacrylate (PMMA). The imaging system records images of the photosensitizer, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS), within the well. AlPcTS was chosen to test the system because of its strong absorption of 635-nm, high fluorescence yield, and relatively long fluorescence lifetime (∼7.5 ns). The results show that the system is capable of resolving 10⁻⁴ M AlPcTS fluorescence as small as 1 mm. The findings of this study contribute to the development of a time-gated imaging system using fluorescence lifetimes. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		In vivo measurement of time-resolved autofluorescence at the human fundus Dietrich Schweitzer, Martin Hammer, Frank Schweitzer, Roswitha Anders, Torsten Doebbecke, Stefan Schenke, E. R. Gaillard, and E. R. Gaillard J. Biomed. Opt. 9, 1214 (2004); http://dx.doi.org/10.1117/1.1806833 \| Cited 23 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract An experimental setup for measurement of time-resolved autofluorescence of the human eye fundus is demonstrated. The method combines laser scanning technique and time-correlated single photon counting. The light source is a laser diode, delivering pulses of about 100 ps duration at a repetition rate of 40 MHz. The excitation wavelength is 446 nm and the cutoff wavelength of fluorescence detection is at 475 nm. The autofluorescence can be determined with a spatial resolution of 80×80 μm² and 25 ps time resolution. The fluorescence decay is optimally approximated by a biexponential model. The dominating lifetime τ1 is shortest in the macula (320 to 380 ps) and reaches 1500 ps in the optic disk. The lifetime τ2 varies between 2 ns and 5 ns, but the spatial distribution is more homogeneous. Respiration of 100% oxygen for 6 min leads to changes in the fluorescence lifetime pointing to detection of coenzymes. Diagrams of lifetime τ2 versus τ1 are well suited for comparison of substances. Such lifetime clusters of a 20 deg macular field of a young healthy subject and of a patient suffering from dry age-related macular degeneration overlap only partially with τ2-τ1 clusters of lipofuscin. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Detection of dual-gene expression in arteries using an optical imaging method Hunter H. Chen, Xiangcan Zhan, Ananda Kumar, Xiangying Du, Holly Hammond, Linzhao Cheng, and Xiaoming Yang J. Biomed. Opt. 9, 1223 (2004); http://dx.doi.org/10.1117/1.1803842 \| Cited 4 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We evaluate the in vivo use of an optical imaging method to detect the vascular expression of green fluorescent protein (GFP) or red fluorescent protein (RFP), and to detect the simultaneous expression of GFP and RFP after transduction into arteries by a dual-promoter lentiviral vector driving their concurrent expression. This method involves using a charge-coupled device camera to detect fluorescence, a fiber optic probe to transmit light, and optical filters to distinguish each marker. In animal models, these vectors are locally delivered to target arteries, whereas the gene for a nonfluorescent cell-surface protein is transduced into contralateral arteries as the sham control. The images show distinct areas of bright fluorescence from GFP and RFP along the target arteries on excitation; no exogenous fluorescence is observed in the controls. Measured signal intensities from arteries transduced with the single- and dual-promoter vectors exceed the autofluorescence signal from the controls. Transgene expression of GFP and RFP in vivo is confirmed with confocal microscopy. We demonstrate the use of an optical imaging method to concurrently detect two distinct fluorescent proteins, potentially permitting the expression of multiple transgenes and their localization in the vasculature to be monitored. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Fast fluorescence lifetime imaging of calcium in living cells A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen J. Biomed. Opt. 9, 1230 (2004); http://dx.doi.org/10.1117/1.1806472 \| Cited 21 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract A fast fluorescence lifetime imaging (FLIM) system is developed that can acquire images at a rate of hundreds of frames per second. The FLIM system is based on a wide-field microscope equipped with a time-gated intensified CCD detector and a pulsed laser. The time-gated detector acquires the signals from two time gates simultaneously and is therefore insensitive to movements of the specimen and photo-bleaching. The system is well suited for quantitative biological FLIM experiments and its performance is evaluated in calcium imaging experiments on beating neonatal rat myocytes. Several calcium sensitive dyes are characterized and tested for their suitability for fast FLIM experiments: Oregon Green Bapta-1 (OGB1), Oregon Green Bapta-2 (OGB2), and Oregon Green Bapta-5N (OGB5N). Overall the sensitivity range of these dyes is shifted to low calcium concentrations when used as lifetime dyes. OGB1 and OGB2 behave very similarly and can be used for FLIM-based calcium imaging in the range 1 to ∼500 nM and OGB5N can be used up to 3 μM. The fast FLIM experiments on the myocytes could be carried out at a 100-Hz frame rate. During the beating of the myocytes a lifetime change of about 20% is observed. From the lifetime images a rest calcium level of about 65 nM is found. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Focused ion beam for microscopy and in situ sample preparation: application on a crustacean digestive system Damjana Drobne, Marziale Milani, Monica Ballerini, Alexis Zrimec, Maja Berden Zrimec, Francesco Tatti, and Kazimir Drašlar J. Biomed. Opt. 9, 1238 (2004); http://dx.doi.org/10.1117/1.1803846 \| Cited 10 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We prove that the focused ion beam (FIB) machine can be used as a microscope and as an in situ cutting device for tissue and cells. For the first time we obtain high-resolution ion images, complemented by electron imaging of different animal tissues both from uncoated and coated samples. In our study, we select the digestive system of Porcellio scaber (isopoda, crustacea) as a test system for FIB microscopy and in situ sample preparation. After the milling operation, some of the ultrastructural elements of hepatopancreatic cells can clearly be recognized. Also, FIB operation reveals significant differences in structural integrity between the apical and basal parts of hepatopancreatic cells, which have not been observed before by classical microscopy techniques. FIB microscopy and in situ sample preparation have advantages over classical microscopy techniques because of: 1. in situ site-specific 2-D sectioning and imaging of subsurface microstructures; 2. no need to embed the sample prior to sectioning; and 3. a wide range of magnifications while imaging the same sample. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution Colin G. Coates, Donal J. Denvir, Noel G. McHale, Keith D. Thornbury, and Mark A. Hollywood J. Biomed. Opt. 9, 1244 (2004); http://dx.doi.org/10.1117/1.1805559 \| Cited 16 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512×512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Nonlinear anisotropic diffusion filtering of three-dimensional image data from two-photon microscopy Philip. J. Broser, R. Schulte, S. Lang, A. Roth Fritjof, Helmchen, J. Waters, Bert Sakmann, and G. Wittum J. Biomed. Opt. 9, 1253 (2004); http://dx.doi.org/10.1117/1.1806832 \| Cited 21 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Two-photon microscopy in combination with novel fluorescent labeling techniques enables imaging of three-dimensional neuronal morphologies in intact brain tissue. In principle it is now possible to automatically reconstruct the dendritic branching patterns of neurons from 3-D fluorescence image stacks. In practice however, the signal-to-noise ratio can be low, in particular in the case of thin dendrites or axons imaged relatively deep in the tissue. Here we present a nonlinear anisotropic diffusion filter that enhances the signal-to-noise ratio while preserving the original dimensions of the structural elements. The key idea is to use structural information in the raw data—the local moments of inertia—to locally control the strength and direction of diffusion filtering. A cylindrical dendrite, for example, is effectively smoothed only parallel to its longitudinal axis, not perpendicular to it. This is demonstrated for artificial data as well as for in vivo two-photon microscopic data from pyramidal neurons of rat neocortex. In both cases noise is averaged out along the dendrites, leading to bridging of apparent gaps, while dendritic diameters are not affected. The filter is a valuable general tool for smoothing cellular processes and is well suited for preparing data for subsequent image segmentation and neuron reconstruction. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Mitigating thermal mechanical damage potential during two-photon dermal imaging Barry R. Masters, Peter T. C. So, Christof Buehler, Nicholas Barry, Jason D. Sutin, William W. Mantulin, and Enrico Gratton J. Biomed. Opt. 9, 1265 (2004); http://dx.doi.org/10.1117/1.1806135 \| Cited 22 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 μm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Detection and diagnosis of oral neoplasia with an optical coherence microscope A. L. Clark, A. Gillenwater, R. Alizadeh-Naderi, A. K. El-Naggar, and R. Richards-Kortum J. Biomed. Opt. 9, 1271 (2004); http://dx.doi.org/10.1117/1.1805558 \| Cited 26 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract The use of high resolution, in vivo optical imaging may offer a clinically useful adjunct to standard histopathologic techniques. A pilot study was performed to investigate the diagnostic capabilities of optical coherence microscopy (OCM) to discriminate between normal and abnormal oral tissue. Our objective is to determine whether OCM, a technique combining the subcellular resolution of confocal microscopy with the coherence gating and heterodyne detection of optical coherence tomography, has the same ability as confocal microscopy to detect morphological changes present in precancers of the epithelium while providing superior penetration depths. We report our results using OCM to characterize the features of normal and neoplastic oral mucosa excised from 13 subjects. Specifically, we use optical coherence and confocal microscopic images obtained from human oral biopsy specimens at various depths from the mucosal surface to examine the optical properties that distinguish normal and neoplastic oral mucosa. An analysis of penetration depths achieved by the OCM and its associated confocal arm found that the OCM consistently imaged more deeply. Extraction of scattering coefficients from reflected nuclear intensity is successful in nonhyperkeratotic layers and shows differentiation between scattering properties of normal and dysplastic epithelium and invasive cancer. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Adaptive spectral apodization for sidelobe reduction in optical coherence tomography images Daniel Marks, P. Scott Carney, and Stephen A. Boppart J. Biomed. Opt. 9, 1281 (2004); http://dx.doi.org/10.1117/1.1806471 \| Cited 5 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Often in imaging systems, the bandpass of the system is not uniform. In temporal coherence imaging methods such as optical coherence tomography, one would like to achieve the most spatially confined impulse response possible with a given source spectrum, minimizing sidelobes that blur adjacent features together. Typically the spectrum of the source is controlled in order to remove sidelobes from the measured interferogram. However, the measured interferogram is not necessarily the best estimate of the scattering density of the object. In this work, a sidelobe supression method is proposed and demonstrated to achieve low sidelobes even with highly nonuniform, non-Gaussian spectra. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Retinal response of Macaca mulatta to picosecond laser pulses of varying energy and spot size William P. Roach, Clarence P. Cain, Drew G. Narayan, Gary D. Noojin, Stephen A. Boppart, Reginald Birngruber, James G. Fujimoto, and Cynthia A. Toth J. Biomed. Opt. 9, 1288 (2004); http://dx.doi.org/10.1117/1.1805554 Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We investigate the relationship between the laser beam at the retina (spot size) and the extent of retinal injury from single ultrashort laser pulses. From previous studies it is believed that the retinal effect of single 3-ps laser pulses should vary in extent and location, depending on the occurrence of laser-induced breakdown (LIB) at the site of laser delivery. Single 3-ps pulses of 580-nm laser energy are delivered over a range of spot sizes to the retina of Macaca mulatta. The retinal response is captured sequentially with optical coherence tomography (OCT). The in vivo OCT images and the extent of pathology on final microscopic sections of the laser site are compared. With delivery of a laser pulse with peak irradiance greater than that required for LIB, OCT and light micrographs demonstrate inner retinal injury with many intraretinal and/or vitreous hemorrhages. In contrast, broad outer retinal injury with minimal to no choriocapillaris effect is seen after delivery of laser pulses to a larger retinal area (60 to 300 μm diam) when peak irradiance is less than that required for LIB. The broader lesions extend into the inner retina when higher energy delivery produces intraretinal injury. Microscopic examination of stained fixed tissues provide better resolution of retinal morphology than OCT. OCT provides less resolution but could be guided over an in vivo, visible retinal lesion for repeated sampling over time during the evolution of the lesion formation. For 3-ps visible wavelength laser pulses, varying the spot size and laser energy directly affects the extent of retinal injury. This again is believed to be partly due to the onset of LIB, as seen in previous studies. Spot-size dependence should be considered when comparing studies of retinal effects or when pursuing a specific retinal effect from ultrashort laser pulses. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Imaging artificial caries under composite sealants and restorations Robert S. Jones, Michal Staninec, and Daniel Fried J. Biomed. Opt. 9, 1297 (2004); http://dx.doi.org/10.1117/1.1805562 \| Cited 23 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Polarization-sensitive optical coherence tomography (PS-OCT) is used to monitor the progression of simulated caries lesions on occlusal surfaces and image the lesions underneath composite sealants. The polarization-sensitive system, recording images in both the parallel and perpendicular axes, is useful for enhancing the image contrast of the artificial caries and minimizing the interference of the strong reflections at surface interfaces. Using the perpendicular-axis signal, the mean reflectivity increase from day 0 to day 14 is 5.1 dB (p<0.01, repeated-measures analysis of variation, Tukey-Kramer). For imaging lesions underneath the sealants, the mean reflectivity of the enamel underneath 250, 500, 750, and 1000 μm of composite is calculated for demineralized and control samples. The artificial lesions can be detected under 750 μm of visibly opaque sealant, with a 5.0-dB difference from the control samples (t-test, p<0.001). Tooth colored sealants allow deeper imaging depth. The artificial lesions could be detected under 1000 μm of sealant, with a 6.6-dB difference from the control samples (t-test, p<0.001). This study demonstrates that PS-OCT can be used to track lesion progression on occlusal surfaces nondestructively with or without sealants. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Design, testing, and clinical studies of a handheld polarized light camera Jessica C. Ramella-Roman, Ken Lee, Scott A. Prahl, and Steven L. Jacques J. Biomed. Opt. 9, 1305 (2004); http://dx.doi.org/10.1117/1.1781667 \| Cited 19 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Polarized light imaging has been used to detect the borders of skin cancer and facilitate assessment of cancer boundaries. A design for an inexpensive handheld polarized camera is presented and clinical images acquired with this prototype are shown. The camera is built with two universal serial bus (USB) color video cameras, a polarizing beamsplitter cube, and a 4× objective lens. Illumination is provided by three white LEDs and a sheet polarizer. Horizontal and vertical linearly polarized reflected images are processed at 7 frames/s and a resulting polarized image is displayed on screen. We compare the performances of cheap USB camera and a 16-bit electronically cooled camera. Dark noise and image repeatability are compared. In both cases, the 16-bit camera outperforms the USB cameras. Despite these limitations, the results obtained with this USB prototype are very satisfactory. Examples of polarized images of lesions taken prior to surgery are presented. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Low-frequency surface wave propagation and the viscoelastic behavior of porcine skin Sean J. Kirkpatrick, Donald D. Duncan, and Li Fang J. Biomed. Opt. 9, 1311 (2004); http://dx.doi.org/10.1117/1.1803843 \| Cited 3 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract A physical model describing the propagation of low-frequency surface waves in relation to the viscoelastic behavior of porcine skin is presented, along with a series of empirical studies testing the performance of the model. The model assumes that the skin behaves as a semi-infinite, locally isotropic, viscoelastic half-space. While the assumption of a semi-infinite body is violated, this violation does not appear to have a significant impact on the performance of the model based on the empirical studies. 1-Hz surface waves in the skin propagate primarily as Rayleigh waves with a wavelength and velocity of approximately 3 m and 3.0 m/s, respectively. The amplitude of the acoustic wave, as measured by tracking the acoustic stress wave-induced shift in a backscattered laser speckle pattern, decreases exponentially with lateral distance from the acoustic source. Using this model of surface wave propagation, the mechanical loss factor or tan δ of the skin is measured to be on the order of 0.14±0.07. The results presented are consistent with earlier works on the propagation of low-frequency acoustic waves in biological tissues, and should serve as a theoretical and empirical basis for using the wave characteristics of propagating surface waves in combination with the mechanical behavior of the tissue for biomechanical studies and for potential diagnostic applications. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Comparison between ultraviolet-visible and near-infrared elastic scattering spectroscopy of chemically induced melanomas in an animal model Ousama M. A’Amar, Ronald D. Ley, and Irving J. Bigio J. Biomed. Opt. 9, 1320 (2004); http://dx.doi.org/10.1117/1.1803845 \| Cited 5 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract The work reported compares elastic scattering spectroscopy (ESS) for diagnosis of pigmented skin lesions in two spectral regions: UV-visible and near infrared (NIR). Given the known strong absorption by melanin in the near-UV to mid-visible range of the spectrum, such a comparison can help determine the optimum wavelength range of ESS for diagnosis of pigmented skin lesions. For this purpose, four South American opossums are treated with dimethylbenz(a)anthracene on multiple dorsal sites to induce both malignant melanomas and benign pigmented lesions. Skin lesions are examined in vivo with ESS using both UV-visible and NIR, with wavelength ranges of 330 to 900 nm and 900 to 1700 nm, respectively. Both portable systems use the same fiber optic probe geometry. ESS measurements are made on the lesions, and spectral differences are grouped by diagnosis from standard histopathological procedure. Both ESS datasets show strong spectral trends with the histopathological assignments, and the data suggest a model for the underlying basis of the spectral distinction between benign and malignant pigmented nevi. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Photoacoustic imaging of blood vessels with a double-ring sensor featuring a narrow angular aperture Roy G. M. Kolkman, Erwin Hondebrink, Wiendelt Steenbergen, Ton G. van Leeuwen, and Frits F. M. de Mul J. Biomed. Opt. 9, 1327 (2004); http://dx.doi.org/10.1117/1.1805556 \| Cited 27 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract A photoacoustic double-ring sensor, featuring a narrow angular aperture, is developed for laser-induced photoacoustic imaging of blood vessels. An integrated optical fiber enables reflection-mode detection of ultrasonic waves. By using the cross-correlation between the signals detected by the two rings, the angular aperture of the sensor is reduced by a factor of 1.9, from 1.5 to 0.8 deg. Consequently, photoacoustic images could be obtained in a manner analogous to the ultrasound B-scan mode. Next, the cross section of artificial blood vessels is visualized by reconstruction of the absorbed energy distribution. Finally, in vivo imaging and the subsequent reconstruction of the absorbed energy distribution is demonstrated for superficial blood vessels in the human wrist. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Determination of optical properties in semi-infinite turbid media using imaging measurements of frequency-domain photon migration obtained with an intensified charge-coupled device Michael Gurfinkel, Tianshu Pan, and Eva M. Sevick-Muraca J. Biomed. Opt. 9, 1336 (2004); http://dx.doi.org/10.1117/1.1803549 \| Cited 6 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Frequency-domain photon migration measurements across the surface of a tissue-mimicking, semi-infinite phantom are acquired via an intensified charge-coupled device (ICCD) detection system and used in conjunction with the diffusion approximation to determine the optical properties. The absorption and reduced scattering coefficients are determined least accurately when relative measurements of average light intensity Idcrel are employed either alone or in a combination with relative modulation amplitude data Iacrel and/or relative phase shift data θ^rel. The absorption and reduced scattering coefficients are found accurate to within 15 and 11%, respectively, of the values obtained from standard single-pixel measurements when θ^rel measurements are employed alone or in combination with Iacrel data. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Detection of differences in oligonucleotide-influenced aggregation of colloidal gold nanoparticles using absorption spectroscopy Mustafa H. Chowdhury, Andrea M. Julian, Craig J. Coates, and Gerard L. Coté J. Biomed. Opt. 9, 1347 (2004); http://dx.doi.org/10.1117/1.1803847 \| Cited 7 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract A rapid, simple, and reproducible assay is described that can be used to detect differences in the ability of oligonucleotides to influence the aggregation of colloidal gold nanoparticles. The aggregation reaction of the gold colloid was monitored through UV-visible absorption spectroscopy. Single isolated colloidal gold particles have a surface plasmon resonance manifested as a single absorbance peak at approximately 520 nm, and aggregated gold complexes develop new red-shifted peaks/shoulders depending on the nature and extent of the aggregated complex. A simple ratiometric study of the area under the single and aggregated plasmon resonance peaks thus gives information about the extent of the aggregation. It is postulated that differences in dynamic flexibility of the oligonucleotides affect their influence on the aggregation state of the gold nanoparticles. The results of this study provide new clues toward unraveling the causes behind the preferential affinity of the Hermes transposable element for certain insertion sites compared to other sequences that also contain recognizable target sites. The technique is robust and thus can potentially be used to study similar questions for numerous transposable elements and target sequences. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Influence of nonhomogeneous distribution of topically applied UV filters on sun protection factors Jürgen Lademann, Andreas Rudolph, Ute Jacobi, Hans-Jürgen Weigmann, Hans Schaefer, Wolfram Sterry, and Martina Meinke J. Biomed. Opt. 9, 1358 (2004); http://dx.doi.org/10.1117/1.1805557 \| Cited 16 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract The aim of the present study is the development of a method to determine quantitatively in vivo the influence of homogeneity of the distribution of sunscreen containing UV filters on the sun protection factor (SPF). The SPF of a sunscreen applied either topically or inside an optical cell (pure or in a solvent) fixed above the skin is determined in vivo. In both cases, in vivo measurements using the erythema formation are carried out. Identical optical parameters of the skin are realized in both experiments. In addition, both in vitro (using tape stripping) and in vivo microscopic measurements are performed to analyze the homogeneity of distribution of the topically applied substances. An SPF of 8 is measured in the experiment applying the UV filters topically, whereas this value increases by a factor of 10 if the same amount of filter substances is distributed homogeneously in solution inside the optical cell. Tape strips removed from skin treated with the sunscreen reflect the inhomogeneous distribution of the topically applied substances on the skin. The direct correlation of homogeneity of distribution with the SPF opens up the possibility to increase the SPF by optimizing the formulation. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Pediatric Vision Screener 1: instrument design and operation David G. Hunter, Deborah S. Nassif, Nadya V. Piskun, Robert Winsor, Boris I. Gramatikov, and David L. Guyton J. Biomed. Opt. 9, 1363 (2004); http://dx.doi.org/10.1117/1.1805560 \| Cited 3 times Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract We develop the Pediatric Vision Screener (PVS) to automatically detect ocular misalignment (strabismus) and defocus in human subjects. The PVS utilizes binocular retinal birefringence scanning to determine when both eyes are aligned, with a theoretical accuracy of <1 deg. The device employs an autoconjugate, bull’s-eye detector-based system to detect focus. The focus and alignment pathways are separated by both wavelength and data acquisition timing. Binocular focus and alignment are detected in rapid alternating sequence, measuring both parameters in both eyes in <0.5 sec. In this work, the theory and design of the PVS are described in detail. With objective, automated measurement of both alignment and focus, the PVS represents a new approach to screening children for treatable eye disease such as amblyopia. © 2004 Society of Photo-Optical Instrumentation Engineers.

Select this Article		Pediatric Vision Screener 2: pilot study in adults Deborah S. Nassif, Nadya V. Piskun, Boris I. Gramatikov, David L. Guyton, and David G. Hunter J. Biomed. Opt. 9, 1369 (2004); http://dx.doi.org/10.1117/1.1805561 \| Cited 1 time Online Publication Date: Nov 22, 2004 Full Text: Read Online (HTML) \| Download PDF
	+ -	Show Abstract Amblyopia is a form of visual impairment caused by ocular misalignment (strabismus) or defocus in an otherwise healthy eye. If detected early, the condition can be fully treated, yet over half of all children with amblyopia under age 5 escape detection. We developed a Pediatric Vision Screener (PVS) to detect amblyopia risk factors. This instrument produces a binocularity score to indicate alignment and a focus score to indicate focus. The purpose of this study is to assess the performance of the PVS by testing adults who were fully cooperative for testing. The study group includes 40 subjects (20 controls, 20 patients) aged 22 to 79 years. 12 patients had constant strabismus (8 to 50^Δ), and eight had variable strabismus (12 to 55^Δ). All controls had binocularity scores >50%. Binocularity was <50% in 11/12 patients. The patient with binocularity >50% had a well-controlled intermittent exotropia and was not at risk for amblyopia. Focus scores were highly sensitive for good focus but not specific. The PVS shows high sensitivity and specificity for detection of strabismus in adults. Future studies will determine whether this performance can be achieved in preschool children, who are at greatest risk for vision loss. © 2004 Society of Photo-Optical Instrumentation Engineers.

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