This PDF file contains the editorial “JBO Letters” for JBO Vol. 10 Issue 03

This PDF file contains the editorial “Infrared and Raman Spectroscopy” for JBO Vol. 10 Issue 03

Infrared spectroscopy, microspectroscopy, and microspectroscopic imaging have been used to probe the composition and physicochemical status of mineral and matrix of bone in normal and diseased tissues using a series of validated parameters that reflect quantitative and qualitative properties. In this review, emphasis is placed on changes in bone's composition and physiochemical status during osteoporosis and the impact of currently used therapeutics on these parameters, although the impact of infrared microscopy in other pathological states is briefly discussed.

In this study we describe a semiautomatic Fourier transform infrared spectroscopic methodology for the analysis of liquid serum samples, which combines simple sample introduction with high sample throughput. The applicability of this new infrared technology to the analysis of liquid serum samples from a cohort of cattle naturally infected with bovine spongiform encephalopathy and from controls was explored in comparison to the conventional approach based on transmission infrared spectroscopy of dried serum films. Artifical neural network analysis of the infrared data was performed to differentiate between bovine spongiform encephalopathy-negative controls and animals in the late stage of the disease. After training of artifical neural network classifiers, infrared spectra of sera from an independent external validation data set were analyzed. In this way, sensitivities between 90 and 96% and specificities between 84 and 92% were achieved, respectively, depending upon the strategy of data collection and data analysis. Based on these results, the advantages and limitations of the liquid sample technique and the dried film approach for routine analysis of biofluids are discussed.

To date, much of our understanding of dentin bonding has been based on investigations performed on sound, healthy dentin. This is not the substrate generally encountered in clinical practice, rather dentists must frequently bond to caries-affected dentin. Because of the extreme complexity and variability of the caries-affected dentin substrate, conventional characterization techniques do not provide adequate information for defining those factors that impact bond formation. Using Fourier-transform infrared imaging, we characterized the inhomogeneities and compositional differences across the length and breadth of the caries-affected dentin/adhesive interface. Differences in mineral/matrix ratio, crystallinity, and collagen organization were noted in the comparison of caries-affected and healthy dentin. As compared to healthy dentin, there were striking differences in depth of demineralization, adhesive infiltration, and degree of conversion at the interface with caries-affected dentin.

The composition of cartilage is predictive of its in vivo performance. Therefore, the ability to assess its primary macromolecular components, proteoglycan (PG) and collagen, is of great importance. In the current study, we hypothesized that PG content and distribution in tissue engineered cartilage could be determined using Fourier-transform infrared imaging spectroscopy (FT–IRIS). The cartilage was grown from chondrocytes within a hollow fiber bioreactor (HFBR) system previously used extensively to study cartilage development. FT–IRIS analysis showed a gradient of PG content, with the highest content in the center near the nutritive fibers and the lowest near the interior surface of the HFBR. Further, we found significantly greater PG content in the region near culture medium inflow (45.0%) as compared to the outflow region (24.7%) (p<0.001). This difference paralleled the biochemically determined glycosaminoglycan difference of 42.6% versus 27.8%. In addition, FT–IRIS-determined PG content at specific positions within the tissue sections correlated with histologically determined PG content (R=0.73, p=0.007). In summary, FT–IRIS determination of PG correlates with histological determination of PG and yields quantitatively similar results to biochemical determination of glycosaminoglycan in developing cartilage.

Both infrared and Raman spectroscopies have the potential to noninvasively estimate the biochemical composition of mammalian cells, although this cannot be unambiguously determined from analysis approaches such as peak assignment or multivariate classification methods. We have developed a fitting routine that determines biochemical composition using basis spectra for the major types of biochemicals found in mammalian cells (protein, DNA, RNA, lipid and glycogen), which is shown to be robust and reproducible. We measured both infrared and Raman spectra of viable suspensions of pairs of nontumorigenic and tumorigenic rat fibroblast cell lines. To model in vivo conditions, we compared nonproliferating, nontumorigenic cells to proliferating, tumorigenic cells. Reproducible differences in biochemical composition were found for both nontumorigenic/tumorigenic cell models, using both spectroscopic techniques. These included an increased fraction of protein and nucleic acids in the tumorigenic cells, with a corresponding decrease in lipid and glycogen fractions. Measurements of each cell type in both the proliferating and nonproliferating states showed that proliferative status was the major determinant of differences in vibrational spectra, rather than tumorigenicity per se. The smallness of the spectral changes associated with tumorgenicity may be due to the subtle nature of the oncogenic change in this system (a single mutant oncogene).

A near infrared (NIR) multispectral imaging method for measuring skin hydration has been applied in a clinical study for estimating skin hydration effects of skin moisturizers and cleansers. The method has been compared to the commercially available, standard electrical methods for evaluating changes in skin hydration based on conductance and capacitance measurements. All of the instrumental methods have been compared to the visual assessment of skin dryness. It has been shown that the NIR imaging method is capable of detecting changes in skin hydration induced by skin moisturizers and cleansers. A large positive hydration change upon treatment by a moisturizing cream was easily detected by all three instrumental methods and by the expert visual assessment of dryness. The imaging technique is rapid, noncontact and noninvasive, and has the additional important advantage of showing the degree of hydration as a function of location, for rapid assessment of change in hydration. There was a clear difference between the instrumental methods when the induced changes were not as great as that from the moisturizing cream. The imaging technique showed more sensitive discrimination between treatments and control, and strong correlation to visual appearance of dryness.

Mid-infrared or Raman spectroscopy together with multivariate data analysis provides a novel approach to clinical laboratory analysis, offering benefits due to its reagent-free nature, the speed of the analysis and the possibility of obtaining a variety of information from one single measurement. We compared mid-infrared and Raman spectra of the sera obtained from 247 blood donors. Partial least squares analysis of the vibrational spectra allowed for the quantification of total protein, cholesterol, high and low density lipoproteins, triglycerides, glucose, urea and uric acid. Glucose (mean concentration: 154 mg/dl) is frequently used as a benchmark for spectroscopic analysis and we achieved a root mean square error of prediction of 14.7 and 17.1 mg/dl for mid-infrared and Raman spectroscopy, respectively. Using the same sample set, comparable sample throughput, and identical mathematical quantification procedures Raman and mid-infrared spectroscopy of serum deliver similar accuracies for the quantification of the analytes under investigation. In our experiments vibrational spectroscopy-based quantification appears to be limited to accuracies in the 0.1 mmol/l range.

We demonstrated the feasibility of Raman spectroscopy for remote temperature monitoring within the aqueous humor of the rabbit eye in vivo. Using a confocal Raman spectroscopy system, Raman spectra from 2580 to 3800 cm–1 were recorded in HPLC-grade water and in the aqueous humor of the rabbit eye under in vivo and ex vivo conditions within a temperature range of 14–34 °C. The ratio between the integrated Raman intensities of two temperature dependent OH-vibrational regions (OH2/OH1) in the spectra of water showed high linear dependence on temperature both in pure water [0.0049(±1.2%)T+0.4522(±0.5%), R²=0.99, n=50, p<0.05], as well as in the rabbit aqueous humor [0.0036(±2.8%)T+0.4966(±0.6%), R²=0.98, n=162, p<0.05] with a high degree of reproducibility and sensitivity (~0.2–0.7 °C). Raman spectroscopy can be used for high resolution and remote monitoring of temperature in the aqueous humor under in vivo conditions.

It has been suggested that spectroscopic analysis of the aqueous humor of the eye could be used to indirectly predict blood glucose levels in diabetics noninvasively. We have been investigating this potential using Raman spectroscopy in combination with partial least squares (PLS) analysis. We have determined that glucose at clinically relevant concentrations can be accurately predicted in human aqueous humor in vitro using a PLS model based on artificial aqueous humor. We have further determined that with proper instrument design, the light energy necessary to achieve clinically acceptable prediction of glucose does not damage the retinas of rabbits and can be delivered at powers below internationally acceptable safety limits. Herein we summarize our current results and address our strategies to improve instrument design.

Tissue modulated Raman spectroscopy was used noninvasively to measure blood glucose concentration in people with type I and type II diabetes with HemoCue fingerstick measurements being used as reference. Including all of the 49 measurements, a Clarke error grid analysis of the noninvasive measurements showed that 72% were A range, i.e., clinically accurate, 20% were B range, i.e., clinically benign, with the remaining 8% of measurements being essentially erroneous, i.e., C, D, or E range. Rejection of 11 outliers gave a correlation coefficient of 0.80, a standard deviation of 22 mg/dL with p<0.0001 for N=38 and places all but one of the measurements in the A and B ranges. The distribution of deviations of the noninvasive glucose measurements from the fingerstick glucose measurements is consistent with the suggestion that there are at least two systematic components in addition to the random noise associated with shot noise, charge coupled device spiking, and human factors. One component is consistent with the known variation of fingerstick glucose concentration measurements from laboratory reference measurements made using plasma or whole blood. A weak but significant correlation between the deviations of noninvasive measurements from fingerstick glucose measurements and the test subject's hemoglobin concentration was also observed.

Surface-enhanced Raman scattering (SERS) has recently been a matter of keen interest from the points of both basic science and applications because by using the SERS effect one can obtain Raman signals even from a single molecule. Immunoassay is one of the most promising fields in the applications of SERS, and the purpose of this review paper is to discuss the potential of SERS in immunoassay. This paper consists of four parts work on the indirect and direct methods of immunoassay via SERS. These methods provide the laboratorial attempts on biomedical diagnostic applications of SERS.

Raman spectroscopy has been well established as a powerful in vitro method for studying biological tissue and diagnosing disease. The recent development of efficient, high-throughput, low-background optical fiber Raman probes provides, for the first time, the opportunity to obtain real-time performance in the clinic. We present an instrument for in vivo tissue analysis which is capable of collecting and processing Raman spectra in less than 2 s. This is the first demonstration that data acquisition, analysis, and diagnostics can be performed in clinically relevant times. The instrument is designed to work with the new Raman probes and includes custom written LabVIEW and Matlab programs to provide accurate spectral calibration, analysis, and diagnosis along with important safety features related to laser exposure. The real-time capabilities of the system were demonstrated in vivo during femoral bypass and breast lumpectomy surgeries. Such a system will greatly facilitate the adoption of Raman spectroscopy into clinical research and practice.

We report the first successful study of the use of Raman spectroscopy for quantitative, noninvasive ("transcutaneous") measurement of blood analytes, using glucose as an example. As an initial evaluation of the ability of Raman spectroscopy to measure glucose transcutaneously, we studied 17 healthy human subjects whose blood glucose levels were elevated over a period of 2–3 h using a standard glucose tolerance test protocol. During the test, 461 Raman spectra were collected transcutaneously along with glucose reference values provided by standard capillary blood analysis. A partial least squares calibration was created from the data from each subject and validated using leave-one-out cross validation. The mean absolute errors for each subject were 7.8%±1.8% (mean±std) with R² values of 0.83±0.10. We provide spectral evidence that the glucose spectrum is an important part of the calibrations by analysis of the calibration regression vectors.

We describe the use of a Teflon®-AF liquid core optical fiber (LCOF) geometry to enhance the collection of Raman scattering from the biochemical creatinine, dissolved in water and in urine. At short integration times, where shot noise is most troublesome, the enhanced signal leads to greater accuracy in estimating the creatinine concentration from the spectrum. At longer integration times, instabilities in the LCOF geometry manifest themselves, and the predictions are the same as or worse than those from standard cuvette-based spectral measurements. Photobleaching of fluorescence from urine is more extensive and more stable in the LCOF as well. Starting from the measured enhancement of a major creatinine Raman band, we calculate the expected ratio of prediction errors obtained using the two geometries, and it agrees closely with the observed ratio. These results indicate that Raman spectroscopy with these Teflon®-AF LCOFs is stable enough for quantitative concentration predictions, accurate to a few percent of the concentration range spanned.

Raman spectroscopy is a powerful diagnostic tool, enabling tissue identification and classification. Mostly, the so-called fingerprint (~400–1800 cm^–1) spectral region is used. In vivo application often requires small flexible fiber-optic probes, and is hindered by the intense Raman signal that is generated in the fused silica core of the fiber. This necessitates filtering of laser light, which is guided to the tissue, and of the scattered light collected from the tissue, leading to complex and expensive designs. Fused silica has no Raman signal in the high wave number region (2400–3800 cm^–1). This enables the use of a single unfiltered fiber to guide laser light to the tissue and to collect scattered light in this spectral region. We show, by means of a comparison of in vitro Raman microspectroscopic maps of thin tissue sections (brain tumors, bladder), measured both in the high wave number region and in the fingerprint region, that essentially the same diagnostic information is obtained in the two wave number regions. This suggests that for many clinical applications the technological hurdle of designing and constructing suitable fiber-optic probes may be eliminated by using the high wave number region and a simple piece of standard optical fiber.

Fourier-transform (FT)-Raman spectroscopy has been used for identification and evaluation of human artherosclerotic lesions, providing biochemical information on arteries. In this work, fragments of human carotid arteries postmortem were analyzed using a FT-Raman spectrometer operating at an excitation wavelength of 1064 nm, power of 200 mW, and spectral resolution of 4 cm^–1. A total of 75 carotid fragments were spectroscopically scanned and FT-Raman results were compared with histopathology. Discriminant analysis using Mahalanobis distance was applied over principal components scores for tissue classification into three categories: nonatherosclerotic, atherosclerotic plaque without calcification and with calcification. Nonatherosclerotic artery, atherosclerotic plaque, and calcified plaque exhibit spectral signatures related to biochemicals presented in each tissue type, such as bands of collagen and elastin (proteins), cholesterol and its esters, and calcium hydroxyapatite and carbonate apatite, respectively. Spectra of nonatherosclerotic artery were then classified into two groups: normal and discrete diffuse thickening of the intima layer (first group) and moderate and intense diffuse thickening of the intima layer (second group). FT-Raman could identify and classify the tissues found in the atherosclerotic process in human carotid in vitro and had the ability to identify alterations to the diffuse thickening of the intima layer and classify it depending on the intensity of the thickening.

Early dental caries detection will facilitate implementation of nonsurgical methods for arresting caries progression and promoting tooth remineralization. We present a method that combines optical coherence tomography (OCT) and Raman spectroscopy to provide morphological information and biochemical specificity for detecting and characterizing incipient carious lesions found in extracted human teeth. OCT imaging of tooth samples demonstrated increased light backscattering intensity at sites of carious lesions as compared to the sound enamel. The observed lesion depth on an OCT image was approximately 290 µm matching those previously documented for incipient caries. Using Raman microspectroscopy and fiber-optic-based Raman spectroscopy to characterize the caries further, spectral changes were observed in PO³₄^- vibrations arising from hydroxyapatite of mineralized tooth tissue. Examination of various ratios of PO³₄^- ν₂, ν₃, ν₄ vibrations against the ν₁ vibration showed consistent increases in carious lesions compared to sound enamel. The changes were attributed to demineralization-induced alterations of enamel crystallite morphology and/or orientation. OCT imaging is useful for screening carious sites and determining lesion depth, with Raman spectroscopy providing biochemical confirmation of caries. The combination has potential for development into a new fiber-optic diagnostic tool enabling dentists to identify early caries lesions with greater sensitivity and specificity.

Craniosynostosis is a severe craniofacial disease where one or more sutures, the fibrous tissue that lies between the cranial bones, fuses prematurely. Some craniosynostosis syndromes are known to be caused by mutations in fibroblast growth factor (FGF) receptors. Mutated FGF receptors are thought to cause constitutive signaling. In this study, heparin acrylic beads released fibroblast growth factor 2 (FGF2) to mimic constitutive signaling by mutated receptors, delivering FGF2 in addition to already existing normal tissue amounts. Fetal day 18.5 mouse sutures were treated with FGF2-soaked beads and cultured in serum free media for 48 h. We have shown previously that this treatment leads to fusion and increased Msx2 expression, but here we use near-infrared Raman imaging to simultaneously examine the mineral components and matrix components of cranial tissue while providing light microscopic spatial information. FGF2-treated mouse sutures show increased v₁ phosphate and v₁ carbonate bandwidths, indicating a slightly chemically modified mineral being rapidly deposited. In addition, FGF2-treated mouse sutures show a marked increase in mineral-to-matrix ratios compared to control mouse sutures, typical of increased mineralization.

This paper describes the properties of vertical-cavity semiconductor devices designed to emit light when driven in forward bias mode and detect optical radiation at wavelengths longer than that of emission when driven in reverse bias mode. The study of this type of devices is motivated by the miniaturization and integration into a single unit of the three functions that a microfluorimeter has to perform, optical pumping, optical detection, and optical filtering of weak light sources. The devices produced can generate fluorescence with a low output power since their emission wavelength can be tuned with that of maximum absorption of the fluorescent dye. We demonstrate also that they can detect low power fluorescence generated in a small volume of concentrated solution of a commercial dye. These devices can find useful application in microanalytical systems such as microfluidic devices or optical biochips.

In a previous study we have shown that correction of peripheral refractive errors can improve the remaining vision of subjects with large central visual field loss. Measuring peripheral refractive errors with traditional methods is often difficult due to low visual acuity and large aberrations. Therefore a Hartmann–Shack sensor has been designed to measure peripheral wave front aberrations in subjects using eccentric viewing. The sensor incorporates an eye tracker and analyzing software designed to handle large wave front aberrations and elliptic pupils. To ensure that the measurement axis is aligned with the direction of the subject's preferred retinal location, a special fixation target has been developed. It consists of concentric rings surrounding the aperture of the sensor together with a central fixation mark along the measurement axis. Some initial measurements on subjects using eccentric viewing have been performed successfully. As a first step in improving the peripheral optics of the eye, the wave front has been used to calculate the eccentric refraction. This refraction has been compared to the refraction found with the PowerRefractor instrument. Measuring the off-axis wave front is a fast way to assess the optical errors in the subject's eccentric viewing angle and to better understand the problems of eccentric correction.

Previous research suggests that the visual impairment of a violet laser is not highly localized on the retina, because the lens absorbs most short-wavelength visible light and partly retransmits it as a diffuse fluorescence at ~500 nm. The present study investigated whether a 405 nm violet diode laser more greatly impairs visual search performance in humans than does a 670 nm red diode laser, depending on target eccentricity. Participants had to locate a square among 15 diamonds spread throughout a visual search display while being exposed to a violet or red laser beam that was either continuous or flickering and presented either on-axis or 33° off-axis. Whereas the continuous on-axis violet and red lasers had comparable effects on search performance when the target was located near the center of the beam, the violet laser disrupted processing of eccentric targets more than did the red laser. The search decrements were reduced for both lasers when the beams were flickered or presented off-axis. Both the bluish appearance and greater spatial spread of effect of the violet laser suggest that the unique impairment caused by a violet laser beam derives from its induced lens fluorescence.

The single cell gel electrophoresis assay is a sensitive, rapid, and visual technique for deoxyribonucleic acid (DNA) strand-break detection in individual mammalian cells, whose application has significantly increased in the past few years. The cells are embedded in agarose on glass slides followed by lyses of the cell membrane. Thereafter, damaged DNA strands are electrophoresed away from the nucleus towards the anode giving the appearance of a comet tail. Nowadays, charge coupled device cameras are attached at optical microscopes for recording the images of the cells, and digital image processing is applied for obtaining quantitative descriptors. However, the conventional software is usually expensive, inflexible and, in many cases, can only provide low-order descriptors based in image segmentation, determination of centers of mass, and Euclidean distances. Associated density functions and centered reduced moments offer an effective and flexible alternative for quantitative analysis of the comet cells. We will show how the position of the center of mass, the lengths and orientation of the main semiaxes, and the eccentricity of such images can be accurately determined by this method.

Surface plasmon resonance (SPR) phase-shift interferometry (PSI) is a novel technique which combines SPR and modified Mach–Zehnder PSI to measure the spatial phase variation caused by biomolecular interactions upon a sensing chip. The SPR-PSI imaging system offers high resolution and high-throughout screening capabilities for microarray DNA hybridization without the need for additional labeling, and provides valuable quantitative information. The SPR-PSI imaging system has an enhanced detection limit of 2.5×10^–7 refraction index change, a long-term phase stability of π/100 in 30 min, and a spatial phase resolution of π/300 with 100×100 µm² detection area. This study successfully demonstrates the label-free observation of 15-mer DNA microarray.

Multiphoton microscopy has evolved into a powerful bioimaging tool in three dimensions. However, the ability to image biological specimens in-depth can be hindered by sample spherical aberration and scattering. These two phenomena can result in the degradation of image resolution and the loss of detected multiphoton signal. In this work, we use the correction collar (for cover glass thickness) associated with a water immersion objective in an attempt to improve multiphoton imaging. In the two samples we examined (human skin and rat tail tendon), we found that while the improvement in image resolution was not visible qualitatively, the measured axial fluorescence or second harmonic generation signal profiles indicate that the use of the correction collar can help to improve the detected multiphoton signals. The maximum increases are 36% and 57% for the skin (sulforhodamine B fluorescence) and tendon (second harmonic generation) specimens, respectively. Our result shows that for in-depth multiphoton imaging, the correction collar may be used to correct for spherical aberration. However, each tissue type needs to be examined to determine the optimal correction collar setting to be used.

Presented is the use of fluorescence lifetime (FLT), anisotropy decay, and associated parameters as differential indicators of cellular activity. A specially designed combination of a frequency mode based time resolved microscope and a picoliter well-per-cell array have been used to perform temporal measurements in individual cells under various biological conditions. Two biological models have been examined: mitogenic activation of peripheral blood mononuclear cells (PBMC) and induction of programmed cell death (apoptosis) in Jurkat T cells (JTC). The FLT of fluorescein stained PBMC was found to increase from 4±0.02 to 4.5±0.025 ns due to mitogenic activation, whereas during apoptosis in fluorescein stained JTC, the FLT remained constant. Notably, the rotational correlation times changed in both models: decreased in PBMC from 2.5±0.08 to 2±0.1 ns, and increased in JTC from 2.1±0.07 to 3.3±0.09 ns. FLT and rotational correlation time were used to calculate the steady state fluorescence anisotropy (FA) which was compared to directly measured FA values. The present study suggests that in addition to bioindication, the said parameters can provide valuable information about cellular mechanisms that may involve complex molecular diffusion dynamics, as well as information about structural changes that a cellular fluorophore undergoes in the course of cell activation.

We propose a novel application of microscopic Förster resonance energy transfer (FRET) to clinical cytological diagnosis based on sensitive measurements of distance changes between fluorescently labeled deoxyribose nucleic acid (DNA) molecules. We have employed the microscopic FRET imaging for investigation of six papillary carcinomas and eight benign cases. In each case the FRET images of 20 cells stained by the AT-specific donor Hoechst 33258 and the GC-specific acceptor 7-aminoactinomycin D were acquired and analyzed by texture analysis. We have not found significant difference of the mean FRET efficiency between the benign and malignant groups. On the other hand, the texture analysis revealed a significant difference of the intranuclear spatial distribution of FRET efficiencies between the benign and malignant groups. The results indicate that despite the similar average distance between the AT- and the GC-rich DNA segments in the papillary carcinomas and the benign cases, the former has more heterogeneous distribution of the AT- and the GC-rich DNA segments in nuclei compared to the benign groups. We have demonstrated that the FRET imaging is a helpful tool for the medical cytological diagnosis of human tumors by giving information on the chromatin topology on the scale below the resolution of conventional optical microscopes.

Fluorescence imaging has been shown to be a potential complement to visual inspection for demarcation of basal cell carcinoma (BCC), which is the most common type of skin cancer. Earlier studies have shown promising results when combining autofluorescence with protoporphyrin IX (Pp IX) fluorescence, induced by application of δ-5-aminolaevulinic acid (ALA). In this work, we have tried to further improve the ability of this technique to discriminate between areas of tumor and normal skin by implementing texture analysis and Fisher linear discrimination (FLD) on bispectral fluorescence data of BCCs located on the face. Classification maps of the lesions have been obtained from histopathologic mapping of the excised tumors. The contrast feature obtained from co-occurrence matrices was found to provide useful information, particularly for the ALA-induced Pp IX fluorescence data. Moreover, the neighborhood average features of both autofluorescence and Pp IX fluorescence were preferentially included in the analysis. The algorithm was trained by using a training set of images with good agreement with histopathology, which improved the discriminability of the validation set. In addition, cross validation of the training set showed good discriminability. Our results imply that FLD and texture analysis are preferential for correlation between bispectral fluorescence images and the histopathologic extension of the tumors.

In this study photomechanical experiments were carried out to examine the relationship between macroscopic mechanical stress and strain gradients within the root dentine structure. Three-dimensional digital photoelasticity was used to study the stress distribution patterns in tooth models, while digital moiré interferometry was used to study the strain gradients within the natural teeth. The stress analysis showed a distinct bending stress distribution, along faciolingual plane in the coronal and cervical regions of the tooth. There was a reduction in bending towards the apical third of the tooth model. The strain analysis displayed strain gradients in the axial (along the long axis of the tooth) and lateral (perpendicular to the long axis of the tooth) directions in dentine. There was a conspicuous reduction in strains from the cervical to the apical third of the root dentine. The root dentine displayed uniform distribution of normal strains. Although there was a steep increase in stresses from the inner core region to the outer surface of an isotropic tooth model, there were more uniform strain gradients in the natural dentine structure. It is apparent from these observations that complex organization of material properties facilitated distinct strain gradients in dentine structure during mechanical functions.

This study represents the first reported use of photodynamic therapy (PDT) for metastatic bone lesions and specifically, as a treatment for spinal metastases. A model of bone metastasis in rat confirmed the efficacy of benzoporphyrin derivative-monoacid-mediated PDT for treating lesions within the spine and appendicular bone. Fluorimetry confirmed the selective accumulation of drug into the tumor(s) at 3 h post-injection. 48 h post-light delivery into the vertebral body of the rat spine loss of bioluminescent signal and histological analyses of sectioned spine confirmed MT-1 tumor cell kill in vivo as previously confirmed in vitro using an established cell viability assay. Porcine vertebrae provided a model comparable to that of human for light propagation and PDT response. Histological examination of vertebrae 48 h post-PDT revealed a necrotic radius of 0.6 cm with an average fluence rate of 4.3 mW/cm². Non-necrotic tissue damage was evident up to 2 cm out from the treatment fiber. Results support the application of PDT to the treatment of primary or metastatic lesions within bone.

The age-related deterioration in bone quality and consequent increase in fracture incidence is an obvious health concern that is becoming increasingly significant as the population ages. Raman spectroscopy with deep-ultraviolet excitation (244 nm) is used to measure vibrational spectra from human cortical bone obtained from donors over a wide age range (34–99 years). The UV Raman technique avoids the fluorescence background usually found with visible and near-infrared excitation and, due to resonance Raman effects, is particularly sensitive to the organic component of bone. Spectral changes in the amide I band at 1640 cm^–1 are found to correlate with both donor age and with previously reported fracture toughness data obtained from the same specimens. These results are discussed in the context of possible changes in collagen cross-linking chemistry as a function of age, and are deemed important to further our understanding of the changes in the organic component of the bone matrix with aging.

In current clinical practice, the initial stage in the detection of pulmonary airway disease is the clinician's interpretation of a bronchoscopy procedure. Due to the nature of this form of detection, diseases are often well advanced upon discovery. This paper proposes a new method for examining the pulmonary airway microcirculatory system via fluorescein imaging techniques similar to those used extensively in ophthalmology. Through the use of a specialized bronchoscopy imaging system we are able to successfully show that fluorescein emission detected in the rabbit trachea exhibits a clear response to inflammatory mediators and thus to changing local vascularity. The clinical implications of this technique are extensive including most importantly the potential use as an evaluation tool for pulmonary disease evaluation and likely other endoscopically accessible organs.

In this paper, a burn color image segmentation and classification system is proposed. The aim of the system is to separate burn wounds from healthy skin, and to distinguish among the different types of burns (burn depths). Digital color photographs are used as inputs to the system. The system is based on color and texture information, since these are the characteristics observed by physicians in order to form a diagnosis. A perceptually uniform color space (L*u*v*) was used, since Euclidean distances calculated in this space correspond to perceptual color differences. After the burn is segmented, a set of color and texture features is calculated that serves as the input to a Fuzzy-ARTMAP neural network. The neural network classifies burns into three types of burn depths: superficial dermal, deep dermal, and full thickness. Clinical effectiveness of the method was demonstrated on 62 clinical burn wound images, yielding an average classification success rate of 82%.

We study theoretically light backscattered by tissues using the radiative transport equation. In particular we consider a two-layered medium in which a finite slab is situated on top of a half space. We solve the one-dimensional problem in which a plane wave is incident normally on the top layer and is the only source of light. The solution to this problem is obtained formally by imposing continuity between the solutions for the upper and lower layers. However, we are interested solely in probing the top layer. Assuming that the optical properties in the lower layer are known, we remove it from the problem yielding a finite slab problem by prescribing an alternate boundary condition. This boundary condition is derived using the theory of Green's functions and is exact. Hence, one needs only to solve the transport equation in a finite slab using this alternate boundary condition. We derive an asymptotic solution for the case when the slab is optically thin. We extend these results to the three-dimensional problem using Fourier transforms. These results are validated by comparisons with numerical solutions for the entire two-layered problem.

A simple empirical method is presented to determine the scattering coefficient µ_s from backscattered polarized images of turbid media. It uses the ratio, pixel by pixel, of two images that are the second and the first backscattered Stokes parameter images Q and I, respectively. Taking this image ratio, then integrating it over the azimuth angle, we get a function depending on the distance from the light entrance point. This function has a maximum. Using Monte Carlo simulations, for a fixed reduced scattering coefficient µ′_s and for an anisotropy factor g varying between 0 and 0.8, it is found a linear relationship between the scattering coefficient µ_s and the inverse of the maximum position of this function.

A simple continuous wave near-infrared algorithm for estimating local hemoglobin oxygen saturation in tissue (%StO₂) is described using single depth attenuation measurements at 680, 720, 760, and 800 nm. Second derivative spectroscopy was used to reduce light scattering effects, chromophores with constant absorption, baseline/instrumentation drift, and movement artifacts. Unlike previous second derivative methods which focused primarily on measuring deoxyhemoglobin concentration; a wide 40 nm wavelength gap used for calculating second derivative attenuation significantly improved sensitivity to oxyhemoglobin absorption. Scaled second derivative attenuation at 720 nm was correlated to in vitro hemoglobin oxygen saturation to generate a %StO₂ calibration curve. The calibration curve was insensitive to total hemoglobin, optical path length, and optical scattering. Measurement error due to normal levels of carboxyhemoglobin, methemoglobin, and water absorption were less than 10 %StO₂ units. Severe methemoglobinemia or edema combined with low blood volume could cause StO₂ errors to exceed 10 StO₂ units. Both a broadband and commercial four-wavelength spectrometer (InSpectraTM) measured %StO₂. The InSpectra tissue spectrometer readily detected limb ischemia on 26 human volunteers for hand, forearm, and leg muscles. A strong linear correlation, r₂>0.93, between StO₂ and microvascular %SO₂ was observed for isolated animal hind limb, kidney, and heart.

Determination of tissue optical properties is fundamental for application of light in either therapeutical or diagnostics procedures. In the present work we implemented a spatially resolved steady-state diffuse reflectance method where only two fibers (one source and one detector) spaced 2.5 mm apart are used for the determination of the optical properties. The method relies on the spectral characteristics of the tissue chromophores (water, dry tissue, and blood) and the assumption of a simple wavelength dependent expression for the determination of the reduced scattering coefficient. Because of the probe dimensions the method is suited for endoscopic measurements. The method was validated against more traditional models, such as the diffusion theory combined with adding doubling for in vitro measurements of bovine muscle. Mean and standard deviation of the absorption coefficient and the reduced scattering coefficient at 630 nm for normal mucosa were 0.87±0.22 cm^–1 and 7.8±2.3 cm^–1, respectively. Cancerous mucosa had values 1.87±1.10 cm^–1 and 8.4±2.3 cm^–1, respectively. These values are similar to data presented by other authors. Blood perfusion was the main variable accounting for differences in the absorption coefficient between the studied tissues.

Dynamic fluorescence images were obtained from a subcutaneous human Kaposi's sarcoma tumor (KS1767) model immediately following the intravenous injection of an integrin-targeting cyanine dye conjugate, Cy5.5-c(KRGDf). The fluorescence images, acquired via an intensified charge-coupled device detection system, were used in conjunction with a pharmacokinetic (PK) model to determine kinetic properties of target binding in the presence and absence of a competitive ligand, free c(KRGDf). The results indicate that the conjugate dye behaves similarly in normal tissue to the free Cy5.5 dye while it possesses increased uptake in tumor tissue. The change in pharmacokinetic parameters obtained from dynamic imaging of Cy5.5-c(KRGDf) after administration of c(KRGDf) as a competitive ligand to the integrin receptor suggests that (i) the increased uptake of Cy5.5-c(KRGDf) is molecularly specific and that (ii) receptor turnover occurs within 24 h. In addition, PK analysis enables quantification of an in vivo c(KRGDf) binding constant attributable to integrin binding. In vivo pharmacokinetic analysis based on rapid and dynamic optical imaging may be potentially useful for evaluating the presence and turnover rate of disease markers that are potential targets of molecular medicine.

Radiofrequency (RF) lesioning in the human brain is one possible surgical therapy for severe pain as well as movement disorders. One obstacle for a safer lesioning procedure is the lack of size monitoring. The aim of this study was to investigate if changes in laser Doppler or intensity signals could be used as markers for size estimation during experimental RF lesioning. A 2 mm in diameter monopolar RF electrode was equipped with optical fibers and connected to a digital laser Doppler system. The optical RF electrode's performance was equal to a standard RF electrode with the same dimensions. An albumin solution with scatterers was used to evaluate the intensity and laser Doppler signal changes during lesioning at 70, 80, and 90 °C. Significant signal changes were found for these three different clot sizes, represented by the temperatures (p<0.05, n=10). The volume, width, and length of the created coagulations were correlated to the intensity signal changes (r=0.88, n=30, p<0.0001) and to the perfusion signal changes (r=0.81, n=30, p<0.0001). Both static and Doppler-shifted light can be used to follow the lesioning procedure as well as being used for lesion size estimation during experimental RF lesioning.

Soft tissues of the body are composite, typically being made up of collagen and elastin fibers with high water contents. The strain measurement in soft tissues has proven to be a difficult task. The digital speckle method, combined with the image processing technique, has many advantages such as full field, noncontact, and real time. We focus on the use of an improved digital speckle correlation method (DSCM) and time-sequence electric speckle pattern interferometry (TSESPI) to noninvasively obtain continual strain measurements on cartilage and vessel tissues. Monoaxial tensile experiments are well designed and performed under constant temperature and the necessary humidity with smart sensors. Mechanical behaviors such as the tensile modulus and Poisson ratio of specimens are extracted based on the deformation information. A comparison of the advantages and the disadvantages of these techniques as well as some problems concerning strain measurements in soft tissues are also discussed.

This PDF file contains the editorial “Introduction to Biophotonics” for JBO Vol. 10 Issue 03

This PDF file contains the editorial “Nanophotonics” for JBO Vol. 10 Issue 03