Induction of heat shock protein (Hsp) expression appears to correlate with a cytoprotective effect in cultured cells and with improved healing of damaged tissues in animal models and in humans. This family of proteins can also serve as indicators of thermal stress in cases of burn injury or surgical procedures that produce heat. Thus, a rapid in vivo readout for induction of Hsp transcription would facilitate studies of Hsp genes and their encoded proteins as mediators of therapeutic effects and as reporters of thermal damage to tissues. We created a transgenic reporter mouse where expression of luciferase is controlled by the regulatory region of the inducible 70 kDa Hsp, and assessed activation of Hsp70 transcription in live animals in response to rapid, high temperature stresses using in vivo bioluminescence imaging (BLI). This model can be used to noninvasively reveal levels of Hsp70 transcription in living tissues, and has utility in studies of the predictive and protective effects of Hsp70 expression, and of various stress responses in tissues.

Oral cancer is an important global health problem. There is an urgent need for improved methods to detect oral cancer and its precursors, because early detection is the best way to reduce oral cancer mortality and morbidity. In this work, we describe simple modifications to a surgical headlight system that enables direct visualization and digital image acquisition from oral tissue in multiple imaging modalities including fluorescence, white-light reflectance, and orthogonal polarization reflectance. Images obtained with the system in-vivo demonstrate that it is an attractive technology to explore for oral cancer screening in low-resource environments where clinical expertise is often unavailable.

We report that the pathologic components present within the atheromatous plaques of ApoE knock-out mice can reflect significant amounts of mid-infrared (mid-IR) light. Furthermore, the reflected light spectra contained the unique signatures of a variety of biologic features including those found in unstable or "vulnerable" plaque. This discovery may represent a unique opportunity to develop a new intravascular diagnostic modality that can detect and characterize sites of atherosclerosis.

Little is known about the relationship between anatomic and functional contrast derived from intrinsic optical signals. In order to address this relationship, finite-element (FEM) forward simulations were compared to diffuse optical spectroscopy (DOS) reflectance measurements obtained from 10 breast tumor patients. Clinical ultrasound images were used to estimate anatomical tumor size and depth for the FEM simulations. Actual DOS-measured tumor absorption could not be matched by forward model simulations when tumor size was constrained to match ultrasound dimensions. However, agreement was achieved when the lesion was viewed as a distribution of optical properties (i.e., an extended target). This result suggests that the spatial extent of optical contrast in breast tumors may be significantly greater than anatomical dimensions reported by standard imaging modalities. Analysis indicates that invasive breast tumors with anatomical dimensions of 1 cm may still be detectable at depths of 30 mm or more (the center of the lesion to the surface of tissue) using DOS in a reflectance geometry.

Heterogeneity in cardiac tissue microstructure is a potential mechanism for the generation and maintenance of arrhythmias. Abnormal changes in fiber orientation increase the likelihood of arrhythmia. We present optical coherence tomography (OCT) as a method to image myofibers in excised intact heart preparations. Three-dimensional (3-D) image sets were gathered from the rabbit right ventricular free wall (RVFW) using a microscope-integrated OCT system. An automated algorithm for fiber orientation quantification in the plane parallel to the wall surface was developed. The algorithm was validated by comparison with manual measurements. Quantifying fiber orientation in the plane parallel to the wall surface from OCT images can be used to help understand the conduction system of the specific sample being imaged.

A fast-scan-rate swept laser for optical coherence tomography (OCT) is suitable to record and analyze a 3-D image volume. However, the whole OCT system speed is limited by data streaming, processing, and storage. In this case, postprocessing is a common technique. Endoscopic clinical applications prefer onsite diagnosis, which requires a real-time technique. Parallel digital signal processors were applied to stream and process data directly from a data digitizer. A real-time system with 20-kHz axial line speed, which was limited only by our swept laser scan rate, was implemented. To couple with the system speed, an endoscope based on an improved 3-D microelectromechanical motor (diameter 1.5 mm, length 9.4 mm) was developed. In vivo 3-D imaging of the human airway was demonstrated.

Silicon phthalocyanine Pc 4 photodynamic therapy (Pc 4-PDT) has emerged as a potentially effective treatment for cutaneous T-cell lymphoma (CTCL). Noninvasive reflectance and fluorescence spectroscopy before, during, and after PDT may provide useful dose metrics and enable therapy to be tailored to individual lesions. We present the design and implementation of a portable bedside spectroscopy system for initial clinical trials of Pc 4-PDT of CTCL. Reflectance and fluorescence spectra were obtained from an early stage CTCL patient throughout the course of the PDT treatment. Preliminary patient data show a significant effect of Pc 4 on the tissue absorption, modest Pc 4 photobleaching, and heterogeneity of Pc 4 within and between the lesions.

This PDF file contains the editorial “Special Section Guest Editorial: Visible Fluorescent Proteins” for JBO Vol. 13 Issue 03

We have witnessed remarkable advances over the past decade in the application of optical techniques to visualize the genetically encoded fluorescent proteins (FPs) in living systems. The imaging of the FPs inside living cells has become an essential tool for studies of cell biology and physiology. FPs are now available that span the visible spectrum from deep blue to deep red, providing a wide choice of genetically encoded fluorescent markers. Furthermore, some FPs have been identified that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells. These additions to the FP toolbox are now being used for some very innovative live-cell imaging applications. Here, we will highlight the characteristics and uses of a few of these exceptional probes. Many different optical methods can be combined with the FPs from marine organisms to provide quantitative measurements in living systems.

The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Typically, the Cerulean and Venus variants of the cyan and yellow FPs are used for FRET studies, but there are limitations to their use. Here, Cerulean and the newly developed monomeric Teal FP (mTFP) are compared as FRET donors for Venus using spectral and fluorescence lifetime measurements from living cells. The results demonstrate that when compared to Cerulean, mTFP has increased brightness, optimal excitation using the standard 458-nm laser line, increased photostability, and improved spectral overlap with Venus. In addition, the two-photon excitation and fluorescence lifetime characteristics are determined for mTFP. Together, these measurements indicate that mTFP is an excellent donor fluorophore for FRET studies, and that its use may improve the detection of interactions involving proteins that are difficult to express, or that need to be produced at low levels in cells.

Förster resonance energy transfer (FRET) is a physical phenomenon used to study molecular interactions in living cells. Changes in the fluorescence lifetime of proteins genetically tagged with a donor fluorophore, such as cyan fluorescent protein or Cerulean, are used to measure energy transfer to a protein tagged with an acceptor fluorophore (yellow fluorescent protein or Venus). Increased transfer efficiency is usually interpreted as closer proximity. Resonance energy transfer is also possible between identical fluorophores. This form of FRET is called energy migration resonance energy transfer (EM-RET). Theoretically, EM-RET should not alter the lifetime or emission spectrum measured from a population of fluorophores. We find a change in the fluorescent lifetime of Cerulean that correlates with energy migration and can result in significant errors when using Cerulean as a donor to measure fluorescence lifetime imaging (FLIM)-FRET efficiencies.

Since its discovery, green fluorescent protein (GFP) and its variants have proven to be a good and convenient fluorescent label for proteins: GFP and other visible fluorescent proteins (VFPs) can be fused selectively to the protein of interest by simple cloning techniques and develop fluorescence without additional cofactors. Among the steadily growing collection of VFPs, several pairs can be chosen that can serve as donor and acceptor fluorophores in Förster resonance energy transfer (FRET) experiments. Among them, the cyan fluorescent proteins (ECFP/Cerulean) and the enhanced yellow fluorescent protein (EYFP) are most commonly used. We show that ECFP and Cerulean have some disadvantages despite their common use: Upon irradiation with light intensities that are commonly used for intensity- and lifetime-based FRET measurements, both the fluorescence intensity and the fluorescence lifetime of ECFP and Cerulean decrease. This can hamper both intensity- and lifetime-based FRET measurements and emphasizes the need for control measurements to exclude these artifacts.

The recycling of G-protein-coupled receptors (GPCR) to the cell surface after internalization plays an important role in the regulation of overall GPCR activity. The angiotensin II type I receptor (AT₁R) belongs to class B GPCRs that recycle slowly back to the cell surface. Previous studies have proposed that Rab11 controls the recycling of AT₁R; however, recent reports show that Rab4, a rapid recycling regulator, co-localizes also with internalized AT₁R. Different from the subcellular co-localization provided by fluorescence microscopy, fluorescence resonance energy transfer (FRET) microscopy provided the spatial relationship of AT₁R with Rab4 and Rab11 in the nanometer-range proximity during the entire course of AT₁R recycling. During the early recycling stage, internalized AT₁Rs were mainly associated with Rab4 in the cytoplasm. During the mid-recycling stage, AT₁Rs were associated with both Rab4 and Rab11 in the perinuclear compartments. However, during the late-recycling stage, AT₁Rs were mainly associated with Rab11, both in the perinuclear compartments and the plasma membrane. Co-immunoprecipitation data confirmed these dynamic associations, which were disrupted by silencing of either the Rab4 or Rab11 gene. Based on these observations, we propose a Rab4 and Rab11 coordinated model for AT₁R recycling.

Förster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ERα) labeled with "wild-type" CFP and YFP is compared with that of ERα labeled with "monomeric" A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced ERα-ERα interaction is similar for ERα labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERα-ERα interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERα-ERα interaction but, when brought close together via ERα-ERα interaction, FP dimerization modestly improves FRET measurement.

Corticotropin releasing hormone receptor (CRHR) and the VT₂ arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the β₂-adrenergic receptor (β₂AR). The VT2R/β₂AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/β₂AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2/β₂AR-chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.

Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk-; cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)–based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system–induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.

Quantum dots (QDs) promise to revolutionize the way fluorescence imaging is used in the cell biology field. The unique fluorescent spectral characteristics, high photostability, low photobleaching, and tight emission spectra of QDs position them above traditional dyes. We will address the ability of water-stabilized QDs to behave as effective fluorescence resonance energy transfer (FRET) donors in cells upon transferrin-receptor–(TFR) mediated endocytosis. Confocal microscopy detects whether donor QD transferrin conjugates transfer energy to acceptor organic fluorophore-transferrin conjugate molecules in endocytic compartments. QDs are shown to be effective FRET donors when internalized into cells via the transferring receptor-mediated endocytic pathway. Upon pairing with the appropriate acceptor dyes, QDs will reduce the laborious data processing that is required to compensate for bleed through contamination between organic dye donor and acceptor pair signals. The QD technology simplifies and expands the use of FRET in the analysis of complex cellular processes that may involve protein organization in intracellular membranes as well as protein-protein interactions.

The construction and application of genetically encoded intracellular calcium concentration ([Ca²⁺]i) indicators has a checkered history. Excitement raised over the creation of new probes is often followed by disappointment when it is found that the initial demonstrations of [Ca²⁺]i sensing capability cannot be leveraged into real scientific advances. Recombinant apo-aequorin cloned from Aequorea victoria was the first Ca²⁺ sensitive protein genetically targeted to subcellular compartments. In the jellyfish, bioluminescence resonance energy transfer (BRET) between Ca²⁺ bound aequorin and green fluorescent protein (GFP) emits green light. Similarly, Ca²⁺ sensitive bioluminescent reporters undergoing BRET have been constructed between aequorin and GFP, and more recently with other fluorescent protein variants. These hybrid proteins display red-shifted spectrums and have higher light intensities and stability compared to aequorin alone. We report BRET measurement of single-cell [Ca²⁺]i based on the use of electron-multiplying charge-coupled-detector (EMCCD) imaging camera technology, mounted on either a bioluminescence or conventional microscope. Our results show for the first time how these new technologies make facile long-term monitoring of [Ca²⁺]i at the single-cell level, obviating the need for expensive, fragile, and sophisticated equipment based on image-photon-detectors (IPD) that were until now the only technical recourse to dynamic BRET experiments of this type.

Proteins from Anthozoa species are homologous to the green fluorescent protein (GFP) from Aequorea victoria but with absorption/emission properties extended to longer wavelengths. HcRed is a far-red fluorescent protein originating from the sea anemone Heteractis crispa with absorption and emission maxima at 590 and 650 nm, respectively. We use ultrasensitive fluorescence spectroscopic methods to demonstrate that HcRed occurs as a dimer in solution and to explore the interaction between chromophores within such a dimer. We show that red chromophores within a dimer interact through a Förster-type fluorescence resonance energy transfer (FRET) mechanism. We present spectroscopic evidence for the presence of a yellow chromophore, an immature form of HcRed. This yellow chromophore is involved in directional FRET with the red chromophore when both types of chromophores are part of one dimer. We show that by combining ensemble and single molecule methods in the investigation of HcRed, we are able to sort out subpopulations of chromophores with different photophysical properties and to understand the mechanism of interaction between such chromophores. This study will help in future quantitative microscopy investigations that use HcRed as a fluorescent marker.

Femtosecond laser pulses in the near-infrared region have potential applications in the imaging and manipulation of intracellular organelles. We report on the manipulation of intracellular organelles by two-photon excitation. The dynamics of green fluorescent protein (GFP)-histone are investigated by two-photon fluorescence recovery after photobleaching (FRAP). Intracellular ablation of fluorescently labeled organelles in living cells is performed by focusing femtosecond laser pulses. We report on the selective marking of individual organelles by using two-photon conversion of a photoconvertible fluorescent protein.

The application of fluorescent proteins in live cells has greatly improved our ability to study molecular mobility, which both reflects molecular function in live cells and reveals the properties of the local environment. Although measuring molecular mobility with fluorescent fusion proteins is powerful and convenient, certain experiments still require exogenous macromolecules to be loaded into cells. Cell viability provides a rough gauge of cellular damage following membrane permeabilization, but it is unknown how permeabilization will affect intracellular mobility. We have used fluorescence correlation spectroscopy to measure the intracellular dynamics of the enhanced green fluorescent protein (EGFP) in living human embryonic kidney (HEK) cells under conditions where the EGFP is either expressed or loaded using streptolysin O (SLO) permeabilization to determine how permeabilization effects mobility. We found that purified EGFP loaded with SLO has the same mobility as the expressed EGFP, while the mobility of the expressed EGFP after SLO permeabilization treatment becomes slightly slower. Our results indicate that SLO permeabilization is often accompanied by the loss of cellular soluble proteins to the surrounding medium, which explains the apparent decrease in diffusion rates following treatment. These measurements are also relevant to the role of molecular crowding in the intracellular mobility of proteins.

The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus effects and in minimizing the overestimation of the molecular brightness. In addition to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady-state conditions, at low expression levels, and in live, unperturbed cells.

Using single-molecule microscopy, we present a method to quantify the number of single autofluorescent proteins when they cannot be optically resolved. This method relies on the measurement of the total intensity emitted by each aggregate until it photobleaches. This strategy overcomes the inherent problem of blinking of green fluorescent proteins. In the case of small protein aggregates, our method permits us to describe the mean composition with a precision of one protein. For aggregates containing a large number of proteins, it gives access to the average number of proteins gathered and a signature of the inhomogeneity of the aggregates' population. We applied this methodology to the quantification of small purified citrine multimers.

Due to their unique optical properties, optical probes, including metal nanoparticles (NPs) and fluorescent dyes, are increasingly used as labeling tools in biological imaging. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) at 750-nm excitation, we recorded intensity and FLIM images from gold NPs (30 nm) and the fluorescent dye Alexa 488 (A488) conjugated with monoclonal ACT-1 antibodies as well as Hoechst 33258 (H258) after incubation with the lymphoma cell line (Karpas-299). From the FLIM images, we can easily discriminate the imaging difference between cells and optical probes according to their distinct fluorescence lifetimes (cellular autofluorescence: 1 to 2 ns; gold NPs: <0.02 ns; A488: 3.5 ns; H258: 2.5 ns). The NP-ACT-1 and A488-ACT-1 conjugates were bound homogeneously on the surface of cells, whereas H258 stained the cell nucleus. We demonstrate that the emission intensity of gold NPs is about ten times stronger than that of the autofluorescence of Karpas-299 cells at the same excitation power. Compared with fluorescent dyes, stronger emission is also observed from gold NPs. Together with their high photostability, these observations suggest that gold NPs are a viable alternative to fluorescent dyes for cellular imaging and cancer diagnosis.

The average fluorescence lifetime of the green fluorescent protein (GFP) in solution is a function of the refractive index of its environment. We report that this is also the case for GFP-tagged proteins in cells. Using time-correlated single-photon counting (TCSPC)–based fluorescence lifetime imaging (FLIM) with a confocal scanning microscope, images of GFP-tagged proteins in cells suspended in different refractive index media are obtained. It is found that the average fluorescence lifetime of GFP decreases on addition of glycerol or sucrose to the media in which the fixed cells are suspended. The inverse GFP lifetime is proportional to the refractive index squared. This is the case for GFP-tagged major histocompatibility complex (MHC) proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP that is located outside the cell membrane. The implications of these findings are discussed with regard to total internal reflection fluorescence (TIRF) techniques where the change in refractive index is crucial in producing an evanescent wave to excite fluorophores near a glass interface. Our findings show that the GFP fluorescence lifetime is shortened in TIRF microscopy in comparison to confocal microscopy.

Cancer progression is commonly accompanied by an altered glucose metabolism. In general, spatially resolved imaging of glucose metabolism and its subtle alterations might provide valuable diagnostic information in vivo. A classical example is positron emission tomography that exploits this feature in obtaining preferential accumulation of fluorescent analog of glucose in tumors, thereby achieving an imaging contrast. We report a novel scaling analysis of glucose metabolism in mammary epithelial (NMuMG) cells by detrended fluctuation analysis of Cerulean (cyan fluorescent protein variant) fluorescence. Fluorescence fluctuations of Cerulean are reasoned to be indicative of dynamic pH changes associated with glucose metabolism. Normal parental cells and the spontaneously transformed (cancerous) NMuMG cells displayed robust scaling exponent that reflects nonrandom fluctuations in Cerulean fluorescence. Acute dependence of cancer cells on glycolysis as compared with normal cells is exploited to yield a statistically significant difference in scaling exponent, thereby providing discrimination between normal and cancer cells in vitro. By careful design of experiments in vivo, the proposed scaling approach might even have diagnostic potential for early detection of cancer lesions in small animal models.

Multiphoton laser scanning microscopy (MPLSM) utilizing techniques such as multiphoton excitation (MPE), second harmonic generation (SHG), and multiphoton fluorescence lifetime imaging and spectral lifetime imaging (FLIM and SLIM, respectively) are greatly expanding the degree of information obtainable with optical imaging in biomedical research. The application of these nonlinear optical approaches to the study of breast cancer holds particular promise. These noninvasive, multidimensional techniques are well suited to image exogenous fluorophores that allow relevant questions regarding protein localization and signaling to be addressed both in vivo and in vitro. Furthermore, MPLSM imaging of endogenous signals from collagen and fluorophores such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD), address important questions regarding the tumor-stromal interaction and the physiologic state of the cell. We demonstrate the utility of multimodal MPE/SHG/FLIM for imaging both exogenous and/or endogenous fluorophores in mammary tumors or relevant 3-D systems. Using SLIM, we present a method for imaging and differentiating signals from multiple fluorophores that can have overlapping spectra via SLIM Plotter—a computational tool for visualizing and analyzing large spectral-lifetime data sets.

Near-infrared spectroscopy (NIRS) is a cerebral monitoring method that noninvasively and continuously measures cerebral hemoglobin oxygenation and the redox state of cytochrome oxidase using highly tissue-permeable near-infrared light. This technique now has wide clinical application, and its usefulness in the measurement of cerebral hemoglobin oxygenation has been confirmed under global cerebral injury and/or hypoxemic hypoxia; however, regional cerebral infarction located far from the monitoring site may not be detected by NIRS. Furthermore, the specificity and accuracy of the measurement of the redox state of cytochrome oxidase remain controversial. We apply NIRS to both animal and clinical investigations. Based on these results, we discuss the significance of the measurement of cerebral hemoglobin oxygenation and cytochrome oxidase in vivo and in clinical medicine. Using our algorithm, cytochrome oxidase signals are unaffected by hemoglobin signals, even when hematocrit values change from 35 to 5% under cardiopulmonary bypass in a dog model. In the clinical study, cytochrome oxidase during surgery is likely to be a good (though not perfect) predictor of postoperative cerebral outcome. NIRS appears to be a promising technology, but additional investigations are required to establish its clinical efficacy and justify its routine use during operative and perioperative periods.

Smoke inhalation injury causes acute airway injury that may result in airway compromise with significant morbidity and mortality. We investigate the ability of high resolution endobronchial optical coherence tomography (OCT) to obtain real-time images for quantitatively assessing regional differences between upper tracheal versus lower tracheal and bronchial airway injury responses to smoke inhalation in vivo using a prototype spectral domain (SLD)-OCT system we constructed, and flexible fiber optic probes. 33 New Zealand White rabbits are intubated and mechanically ventilated. The treatment groups are exposed to inhaled smoke. The OCT probe is introduced through the endotracheal tube and maintained in place for 5 to 6 h. Images of airway mucosa and submucosa are obtained at baseline and at specified intervals postexposure. Starting within less than 15 min after smoke inhalation, there is significant airway thickening in the smoke-exposed animals. This is maintained over 5 h of imaging studies. The lower tracheal airway changes, correlating closely with carboxyhemoglobin levels, are much greater than upper tracheal changes. Significant differences are seen in lower trachea and bronchi after acute smoke inhalation compared to upper trachea as measured in vivo by minimally invasive OCT. OCT is capable of quantitatively detecting regional changes in airway swelling following inhalation injury.

An optical coherence tomography (OCT) prediction algorithm is designed and tested on a data set of sample images (taken from vegetables and porcine tissues) to demonstrate proof of concept. Preprocessing and classification of data are fully automated, at a rate of 60,000 A-scans/min on a standard computer and can be considered to deliver in near real-time. A data set consisting of nine groups was classified correctly in 82% of cases after cross-validation. Sets of fewer groups reach higher rates. The algorithm is able to distinguish groups with strong visual similarity among several groups of varying resemblance. Surface recognition and normalizing to the surface are essential for this approach. The mean divided by the standard deviation is a suitable descriptor for reducing a set of surface normalized A-scans. The method enables grouping of separate A-scans and is therefore straightforward to apply on 3-D data. OCT data can reliably be classified using principal component analysis combined with linear discriminant analysis. It remains to be shown whether this algorithm fails in the clinical setting, where interpatient variation can be greater than the deviations that are investigated as a disease marker.

Intravascular optical coherence tomography (OCT) has been proven a powerful diagnostic tool for cardiovascular diseases. However, the optical mechanism for the qualitative observations are still absent. We address the fundamental issues that underlie the tissue characterization of OCT images obtained from coronary arteries. For this, we investigate both the attenuation and the backscattering properties of different plaque components of postmortem human cadaver coronary arteries. The artery samples are examined both from lumen surface using a catheter and from transversely cut surface using an OCT microscope, where OCT images could be matched to histology exactly. Light backscattering coefficient μb and attenuation coefficients μt are determined for three basic plaque types based on a single-scattering physical model: calcification (μ_b=4.9±1.5 mm^-1, μ_t=5.7±1.4 mm^-1), fibers (μ_b=18.4±6.4 mm^-1, μ_t=6.4±1.2 mm^-1), and lipid pool (μ_b=28.1±8.9 mm^-1, μ_t=13.7±4.5 mm^-1). Our results not only explain the origins of many qualitative OCT features, but also show that combination of backscattering and attenuation coefficient measurements can be used for contrast enhancing and better tissue characterization.

We describe how phase-modulation laser interference microscopy and wavelet analysis can be applied to noninvasive nonstained visualization and study of the structural and dynamical properties of living cells. We show how phase images of erythrocytes can reveal the difference between various erythrocyte forms and stages of hemolysis and how phase images of neurons reveal their complex intracellular structure. Temporal variations of the refractive index are analyzed to detect cellular rhythmic activity on different time scales as well as to uncover interactions between the cellular processes.

Since 1978 in vitro fertilization (IVF) procedures have resulted in the birth of over 3 million babies. Yet in 2005, IVF procedures had a live birth rate of only 34%, with 32% of these births resulting in multiple pregnancies. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. The predominantly accepted noninvasive viability markers for embryos created by IVF are (1) number of cells at specific time points during development and (2) overall morphology of the embryo. Currently, it is difficult to count the number of cells beyond the eight-cell stage noninvasively. We report a nontoxic cell-counting method capable of counting cell numbers ranging from 8 to 26 in live mouse embryos. This method is derived from the fusion of differential interference contrast and optical quadrature microscopy and is verified by epifluorescence images of Hoechst-stained nuclei. The phase-subtraction cell-counting method is the first accurate, nontoxic technique to count cells through the morula stage in mouse embryos and may enhance the use of cell number as a viability marker if adopted for use with human embryos in the IVF clinic.

Biochips have been an advanced technology for biomedical applications since the end of the 20th century. Optical detection systems have been a very important tool in biochip analysis. Microscopes are often inadequate for high resolution and big view-area detection of microarray chips, thus some new optical instruments are required. In this work, a novel digital imaging scanning system with dark-field irradiation is developed for some biomedical applications for microarray chips, characterized by analyzing genes and proteins of clinical samples with high specific, parallel, and nanoliter samples. The novel optical system has a high numerical aperture (NA=0.72), a long working distance (w_d>3.0 mm), an excellent contrast and signal-to-noise ratio, a high resolving power close to 3 μm, and an efficiency of collected fluorescence more than two-fold better than that of other commercial confocal biochip scanners. An edge overlap algorithm is proposed for the image restructure of free area detection and correcting scanning position errors to a precision of 1 pixel. A novel algorithm is explored for recognizing the target from the scanning images conveniently, removing noise, and producing the signal matrix of biochip analysis. The digital imaging scanning system is equally as good for the detection of enclosed biochips as it is for the detection of biological samples on a slide surface covered with a glass cover slip or in culture solution. The clinical bacteria identification and serum antibody detection of biochips are described.

The micropallet array system uses a pulsed laser to release pallets tens of microns to hundreds of micrometers in size from a larger array, enabling selective isolation of single cells adherent to the pallets. We characterize the laser-based release of pallets with respect to pallet array and laser parameters. The threshold laser energy required for pallet release increases linearly with the area of the pallet in contact with the underlying glass substrate. The spacing of the pallets within an array as well as the thickness or height of the pallet does not impact the energy required to release a pallet. Delivery of multiple laser pulses decreases the energy/pulse required for pallet release when the pallets were 100 µm or greater on a side. In addition to the square pallets, complex structures such as cantilevers and spirals could be released without damage using the pulsed laser. Identification of the pallet-array variables influencing the energy required for pallet release as well as strategies to minimize this energy will prove critical in optimizing the release of pallets with cells on the arrays.

Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.

Fluorescence measurements have been used to track the dosimetry of photodynamic therapy (PDT) for many years, and this approach can be especially important for treatments with aminolevulinic-acid-induced protoporphyrin IX (ALA-PpIX). PpIX photobleaches rapidly, and the bleaching is known to be oxygen dependent, and at the same time, fractionation or reduced irradiance treatments have been shown to significantly increase efficacy. Thus, in vivo measurement of either the bleaching rate and/or the total bleaching yield could be used to track the deposited dose in tissue and determine the optimal treatment plans. Fluorescence in rat esophagus and human Barrett's esophagus are measured during PDT in both continuous and fractionated light delivery treatment, and the bleaching is quantified. Reducing the optical irradiance from 50 to 25 mW/cm did not significantly alter photobleaching in rat esophagus, but fractionation of the light at 1-min on and off intervals did increase photobleaching up to 10% more (p value=0.02) and up to 25% more in the human Barrett's tissue (p value<0.001). While two different tissues and two different dosimetry systems are used, the data support the overall hypothesis that light fractionation in ALA-PpIX PDT esophageal treatments should have a beneficial effect on the total treatment effect.

Photodynamic therapy (PDT) is a promising cancer treatment. PDT uses the affinity of photosensitizers to be selectively retained in malignant tumors. When tumors, pretreated with the photosensitizer, are irradiated with visible light, a photochemical reaction occurs and tumor cells are destroyed. Oxygen molecules in the metastable singlet delta state O₂(¹Δ) are believed to be the species that destroys cancerous cells during PDT. Monitoring singlet oxygen produced by PDT may lead to more precise and effective PDT treatments. Our approach uses a pulsed diode laser-based monitor with optical fibers and a fast data acquisition system to monitor singlet oxygen during PDT. We present results of in vitro singlet oxygen detection in solutions and in a rat prostate cancer cell line as well as PDT mechanism modeling.

An ultrasound technique to measure the spatial and temporal behavior of the laser-induced cavitation bubble is introduced. The cavitation bubbles were formed in water and in gels using a nanosecond pulsed Nd:YAG laser operating at 532 nm. A focused, single-element, 25-MHz ultrasound transducer was employed both to detect the acoustic emission generated by plasma expansion and to acoustically probe the bubble at different stages of its evolution. The arrival time of the passive acoustic emission was used to estimate the location of the cavitation bubble's origin and the time of flight of the ultrasound pulse-echo signal was used to define its spatial extent. The results of ultrasound estimations of the bubble size were compared and found to be in agreement with both the direct optical measurements of the stationary bubble and the theoretical estimates of bubble dynamics derived from the well-known Rayleigh model of a cavity collapse. The results of this study indicate that the proposed quantitative ultrasound technique, capable of detecting and accurately measuring laser-induced cavitation bubbles in water and in a tissue-like medium, could be used in various biomedical and clinical applications.

The level of exhaled carbon monoxide (eCO) is considered a marker of oxidative stress in diabetes. Previous findings indicated that eCO levels correlated with blood glucose level. The aim of this work was to apply and compare two independent analyzing methods for eCO after oral glucose administration. Glycemia, eCO, and exhaled hydrogen were measured before and after oral administration of glucose. Six healthy nonsmoking volunteers participated. For eCO analysis, we used two methods: a commercially available electrochemical sensor, and a high-precision laser spectrometer developed in our laboratory. The precision of laser-spectroscopic eCO measurements was two orders of magnitude better than the precision of the electrochemical eCO measurement. eCO levels measured by laser spectrometry after glucose administration showed a decrease of 4.1%±1.5% compared to the baseline (p<0.05). Changes in the eCO measured by the electrochemical sensor were not significant (p=0.08). Exhaled hydrogen levels increased by 40% within the first 10 min after glucose administration (p<0.05). The previous finding that the glycemia increase after glucose administration was associated with a significant increase in eCO concentrations was not confirmed. We propose that previous eCO measurements with electrochemical sensors may have been affected by cross sensitivity to hydrogen.

The purpose of this study is to apply near-infrared (NIR) Raman spectroscopy and classification and regression tree (CART) techniques for identifying molecular changes of tissue associated with cancer transformation. A rapid-acquisition NIR Raman system is utilized for tissue Raman spectroscopic measurements at 785-nm excitation. 73 gastric tissue samples (55 normal, 18 cancer) from 53 patients are measured. The CART technique is introduced to develop effective diagnostic algorithms for classification of Raman spectra of different gastric tissues. 80% of the Raman dataset are randomly selected for spectral learning, while 20% of the dataset are reserved for validation. High-quality Raman spectra in the range of 800 to 1800 cm-1 are acquired from gastric tissue within 5 s. The diagnostic sensitivity and specificity of the learning dataset are 90.2 and 95.7%; and the predictive sensitivity and specificity of the independent validation dataset are 88.9 and 92.9%, respectively, for separating cancer from normal. The tissue Raman peaks at 875 and 1745 cm-1 are found to be two of the most significant features to discriminate gastric cancer from normal tissue. NIR Raman spectroscopy in conjunction with the CART technique has the potential to provide an effective and accurate diagnostic means for cancer detection in the gastric system.

Monte Carlo (MC) modeling of photon transport in tissues is generally performed using simplified functions that only approximate the angular scattering properties of tissue constituents. However, such approximations may not be sufficient for fully characterizing tissue scatterers such as cells. Finite-difference time-domain (FDTD) modeling provides a flexible approach to compute realistic tissue phase functions that describe probability of scattering at different angles. We describe a computational framework that combines MC and FDTD modeling, and allows random sampling of scattering directions from FDTD phase functions. We carry out simulations to assess the influence of incorporating realistic FDTD phase functions on modeling spectroscopic reflectance signals obtained from normal and precancerous epithelial tissues. Simulations employ various fiber optic probe designs to analyze the sensitivity of different probe geometries to FDTD-generated phase functions. Combined MC/FDTD modeling results indicate that the form of the phase function used is an important factor in determining the reflectance profile of tissues, and detected reflectance intensity can change up to ~30% when a realistic FDTD phase function is used instead of an approximating function. The results presented need to be taken into account when developing photon propagation models or implementing inverse algorithms to extract optical properties from measurements.

We explore the use of Monte-Carlo-model-based approaches for the analysis of fluorescence and diffuse reflectance spectra measured ex vivo from breast tissues. These models are used to extract the absorption, scattering, and fluorescence properties of malignant and nonmalignant tissues and to diagnose breast cancer based on these intrinsic tissue properties. Absorption and scattering properties, including β-carotene concentration, total hemoglobin concentration, hemoglobin saturation, and the mean reduced scattering coefficient are derived from diffuse reflectance spectra using a previously developed Monte Carlo model of diffuse reflectance. A Monte Carlo model of fluorescence described in an earlier manuscript was employed to retrieve the intrinsic fluorescence spectra. The intrinsic fluorescence spectra were decomposed into several contributing components, which we attribute to endogenous fluorophores that may present in breast tissues including collagen, NADH, and retinol/vitamin A. The model-based approaches removes any dependency on the instrument and probe geometry. The relative fluorescence contributions of individual fluorescing components, as well as β-carotene concentration, hemoglobin saturation, and the mean reduced scattering coefficient display statistically significant differences between malignant and adipose breast tissues. The hemoglobin saturation and the reduced scattering coefficient display statistically significant differences between malignant and fibrous/benign breast tissues. A linear support vector machine classification using (1) fluorescence properties alone, (2) absorption and scattering properties alone, and (3) the combination of all tissue properties achieves comparable classification accuracies of 81 to 84% in sensitivity and 75 to 89% in specificity for discriminating malignant from nonmalignant breast tissues, suggesting each set of tissue properties are diagnostically useful for the discrimination of breast malignancy.

The temperature dependence (30 to 40°C) of near-infrared spectra (500 to 1100 nm) of whole human blood, including red blood cells with intact physiological function, is investigated. Previous studies have focused on hemoglobin solutions, but the operation of red blood cells is critically dependent on intact cell membranes to perform normal oxygen transport and other physiological functions. Thus measurements of whole blood are more directly related to changes that occur in vivo. In addition to the response of hemoglobin to temperature in the spectra, a temperature response from water in the plasma is also detected. The temperature response of the water absorption at 960 nm is approximately ten times smaller than the temperature response of the oxyhemoglobin component in the blood at 610 nm. However, it is the most significant temperature effect between 800 and 1000 nm. This work will aid the precision and understanding of full spectrum near-infrared measurements on blood.

A multidetector, continuous wave, near-infrared spectroscopy (NIRS) system is developed to examine whether the hemodynamics of the scalp and brain in adults contain significant layer-like hemodynamic trends. NIRS measurements are made using contrasting geometries, one with four detectors equidistant from a source 33 mm away, and one with detectors collinear with the source (5 to 33 mm away). When NIRS time series are acquired over the prefrontal cortex from resting adults using both geometries, variations among the time series are consistent with a substantially homogeneous two-layer model (p<0.001) and inconsistent with one dominated by heterogeneities. Additionally, when time series measured 5 mm from the source are subtracted from corresponding 33-mm signals via a least-squares algorithm, 60% of the hemoglobin changes are on average removed. These results suggest that hemodynamic trends present in the scalp can contribute significantly to NIRS measurements, and that attempts to reduce this noise by subtracting a simultaneous near-channel measurement using a two-layer model are justified. Such subtractions are then performed on NIRS measurements from two stimulus protocols. For systemic stimulations (Valsalva maneuver), the subtraction cancels the hemodynamic response, as desired. For localized stimulation of the occipital lobe (viewing a flickering pattern), the subtraction isolated a stimulus-correlated hemodynamic feature from background noise.

The efficacy of fluorescence spectroscopy to detect squamous cell carcinoma is evaluated in an animal model following laser excitation at 442 and 532 nm. Lesions are chemically induced with a topical DMBA application at the left lateral tongue of Golden Syrian hamsters. The animals are investigated every 2 weeks after the 4th week of induction until a total of 26 weeks. The right lateral tongue of each animal is considered as a control site (normal contralateral tissue) and the induced lesions are analyzed as a set of points covering the entire clinically detectable area. Based on fluorescence spectral differences, four indices are determined to discriminate normal and carcinoma tissues, based on intraspectral analysis. The spectral data are also analyzed using a multivariate data analysis and the results are compared with histology as the diagnostic gold standard. The best result achieved is for blue excitation using the KNN (K-nearest neighbor, a interspectral analysis) algorithm with a sensitivity of 95.7% and a specificity of 91.6%. These high indices indicate that fluorescence spectroscopy may constitute a fast noninvasive auxiliary tool for diagnostic of cancer within the oral cavity.

Ex vivo multiphoton imaging is used to characterize rabbit corneal wound healing after conductive keratoplasty (CK) procedures. CK is performed on the right eyes from eight New Zealand albino rabbits while the left eyes are punctured by a keratoplast tip without energy application. Rabbits are humanely sacrificed 1 day, 1, 2, and 4 weeks after the CK procedure. Eye balls are enucleated and placed on the microscope for multiphoton imaging. Multiphoton imaging reveals damage of corneal epithelium and stroma caused by the CK procedure and the subsequent wound healing process can be followed without histological procedures. Multiphoton excited autofluorescence images demonstrate that re-epithelilialization is accomplished within 1 week in both CK and control groups. However, epithelial hyperplasia is observed in CK corneas. In addition, stromal wounds in the control group become inconspicuous within 1 week while obvious wounds still exist in CK corneas for at least 4 weeks. Postconductive keratoplasty corneal damage and wound healing can be characterized by multiphoton microscopy without histological procedures. Our results suggest that multiphoton microscopy has potential in the clinical evaluation of corneal damage due to refractive surgery, and can be used to study and reduce the unwanted side effects of these procedures.

Miniature endoscopic microscopes, with subcellular imaging capabilities, will enable in vivo detection of molecularly-targeted fluorescent probes for early disease detection. To optimize a dual-axis confocal microscope (DACM) design for this purpose, we use a tabletop instrument to determine the ability of this technology to perform optical sectioning deep within tissue. First, we determine how tissue scattering deteriorates the diffraction-limited transverse and vertical responses in reflectance imaging. Specifically, the vertical response of a DACM to a plane reflector is measured at various depths in a scattering phantom and compared with diffraction theory and Monte Carlo scattering simulations. Similarly, transverse line scans across a knife-edge target are performed at various depths in a scattering phantom. Second, as a practical demonstration of deep-tissue fluorescence microscopy that corroborates the findings from our scattering experiments, 3-D fluorescence images are obtained in thick human gastrointestinal mucosal specimens. Our results demonstrate efficient rejection of scattered light in a DACM, which enables deep optical sectioning in tissue with subcellular resolution that can distinguish between normal and premalignant pathologies.

Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.

Many naturally occurring materials are autofluorescent, a property that can reduce the discriminative ability of fluorescence methods, sometimes to the point where they cannot be usefully applied. Shifting from the spectral to the temporal domain, it is possible to discriminate fluorophores on the basis of their fluorescence decay lifetime. Luminophores with sufficiently long lifetimes can be discriminated from short-lived autofluorescence using time-gated luminescence (TGL). This technique relies upon the application of a brief excitation pulse followed by a resolving period to permit short-lived autofluorescence to decay, after which detection is enabled to capture persistent emission. In our studies, a high-power UV LED was mounted in the filter capsule of an Olympus BX51 microscope to serve as the excitation source. The microscope was fitted with an Andor DV885 electron-multiplying CCD (EM-CCD) camera with the trigger input synchronized to UV LED operation. Giardia lamblia cysts labeled with the europium chelate BHHST were analyzed against an autofluorescent background with the TGL microscope. The EM-CCD camera captured useful TGL images in real time with a single exposure cycle. With 4x frame averaging, images acquired in TGL mode showed a 30-fold improvement in SNR compared with conventional fluorescence microscopy.

A new method is developed to perform local measurements of fluorophore excited state lifetimes in turbid media without collecting the fluorescence emission. The method is based on a pump-probe approach where a first laser pulse excites the dye and then a second laser pulse is used for photoacoustic probing of the transient absorption. The photoacoustic response generated by the probe pulse is recorded by an ultrasound receiver. Repeating the measurement for increasing pump-probe time delays yields a series of photoacoustic signals that are used to extract the lifetime of the excited state. The method is validated by measuring the lifetime of an oxygen sensitive dye solution at different concentrations of dissolved oxygen. The dye is pumped with a 532-nm pulsed laser and the transient absorption at 740 nm is probed using a second pulsed laser system. The photoacoustic-based results are in close agreement with those obtained from time-dependent fluorescent measurements. The method can be extended to photoacoustic lifetime imaging by using a receiver array instead of a single receiver. Potential applications of this method include tissue oxygen imaging for cancer diagnostics and mapping molecular events such as resonant energy transfer and ion collisions in a biological environment.

Photothermal therapy is a noninvasive, targeted, laser-based technique for cancer treatment. During photothermal therapy, light energy is converted to heat by tumor-specific photoabsorbers. The corresponding temperature rise causes localized cancer destruction. For effective treatment, however, the presence of photoabsorbers in the tumor must be ascertained before therapy and thermal imaging must be performed during therapy. This study investigates the feasibility of guiding photothermal therapy by using photoacoustic imaging to detect photoabsorbers and to monitor temperature elevation. Photothermal therapy is carried out by utilizing a continuous wave laser and metal nanocomposites broadly absorbing in the near-infrared optical range. A linear array-based ultrasound imaging system is interfaced with a nanosecond pulsed laser to image tissue-mimicking phantoms and ex-vivo animal tissue before and during photothermal therapy. Before commencing therapy, photoacoustic imaging identifies the presence and spatial location of nanoparticles. Thermal maps are computed by monitoring temperature-induced changes in the photoacoustic signal during the therapeutic procedure and are compared with temperature estimates obtained from ultrasound imaging. The results of our study suggest that photoacoustic imaging, augmented by ultrasound imaging, is a viable candidate to guide photoabsorber-enhanced photothermal therapy.

Artificially created demineralized and remineralized carious lesions on the root and enamel of human teeth were examined by photothermal radiometry (PTR) and modulated luminescence (LUM). Fourteen extracted human teeth were used and a lesion was created on a 1 mm×4 mm rectangular window, spanning root to enamel, using a lactic acid-based acidified gel to demineralize the tooth surface. The lesion was then exposed to a remineralization solution. Each sample was examined with PTR/LUM on the root and enamel before and after treatment at times from 1 to 10 (5 on root) days of demineralization and 2 to 10 days of remineralization. Ten-day (5 on root) demineralized samples were remineralized. After completing all the experiments, transverse microradiography (TMR) analysis was performed to compare and correlate the PTR/LUM signals to the depth of lesions and mineral losses. The PTR and LUM amplitudes and phases showed gradual and consistent changes with treatment time. In this study, TMR showed good correlation coefficients with PTR and LUM. It was also found that the length of the treatment time did not correlate very well to any technique, PTR/LUM or TMR, which implies a significant degree of inhomogeneity of the demireralization and remineralization rates in each and every tooth.

Cancellous bone is a widespread structure in a creatural body, for instance, in the femoral head and spondyle. The damage evolution and crack growth of cattle cancellous bone were studied under three-point-bending load conditions. A series of speckle images with deformation information surrounding the crack tip were recorded, and the full-field displacement distributions were obtained at different loading levels by means of digital speckle correlation method (DSCM). Characterizations of the damage deformation and fracture of cancellous bone were analyzed. These results provide some useful information for studying the fracture behavior of cancellous bone.

Spectacle-mounted telescopic systems are prescribed for individuals with visual impairments. Bioptic telescopes are typically mounted toward the top of the spectacle lens (or above the frame) with the telescope eyepiece positioned above the wearer's pupil. This allows the wearer to use up and down head tilt movements to quickly alternate between the unmagnified wide view (through the carrier lens) and the magnified narrow field of view (available through the eyepiece). Rejection of this visual aid has been attributed mainly to its appearance and to the limited field of view through the smaller Galilean designs. We designed a wide-field Keplerian telescope that is built completely within the spectacle lens. The design uses embedded mirrors inside the carrier lens for optical pathway folding, and conventional lenses or curved mirrors for magnification power. The short height of the ocular, its position, and a small tilt of the ocular mirror enable the wearer to simultaneously view the magnified field above the unmagnified view of the uninterrupted horizontal field. These features improve the cosmetics and utility of the device. The in-the-lens design allows the telescope to be mass produced as a commodity ophthalmic lens blank that can be surfaced to include the wearer's spectacle prescription.

This PDF file contains the errata for “JBO Vol. 13 Issue 03 Paper 2937889” for JBO Vol. 13 Issue 03

This PDF file contains the errata for “JBO Vol. 13 Issue 03 Paper 2943294” for JBO Vol. 13 Issue 03

This PDF file contains the errata for “JBO Vol. 13 Issue 03 Paper 2953560” for JBO Vol. 13 Issue 03