Fluorescence microscopy allows real-time monitoring of optical molecular probes for disease characterization, drug development, and tissue regeneration. However, when a biological sample is thicker than 1 mm, intense scattering of light would significantly degrade the spatial resolution of fluorescence microscopy. In this paper, we develop a fluorescence microtomography technique that utilizes the Monte Carlo method to image fluorescence reporters in thick biological samples. This approach is based on an l0-regularized tomography model and provides an excellent solution. Our studies on biomimetic tissue scaffolds have demonstrated that the proposed approach is capable of localizing and quantifying the distribution of optical molecular probe accurately and reliably.

We present a phase fluctuation calibration method for polarization-sensitive swept-source optical coherence tomography (PS-SS-OCT) using continuous polarization modulation. The method uses a low-voltage broadband polarization modulator driven by a synchronized sinusoidal burst waveform rather than an asynchronous waveform, together with the removal of the global phases of the measured Jones matrices by the use of matrix normalization. This makes it possible to average the measured Jones matrices to remove the artifact due to the speckle noise of the signal in the sample without introducing auxiliary optical components into the sample arm. This method was validated on measurements of an equine tendon sample by the PS-SS-OCT system.

Intrigued by our recent finding that the nuclear refractive index is significantly increased in malignant cells and histologically normal cells in clinical histology specimens derived from cancer patients, we sought to identify potential biological mechanisms underlying the observed phenomena. The cell cycle is an ordered series of events that describes the intervals of cell growth, DNA replication, and mitosis that precede cell division. Since abnormal cell cycles and increased proliferation are characteristic of many human cancer cells, we hypothesized that the observed increase in nuclear refractive index could be related to an abundance or accumulation of cells derived from cancer patients at a specific point or phase(s) of the cell cycle. Here we show that changes in nuclear refractive index of fixed cells are seen as synchronized populations of cells that proceed through the cell cycle, and that increased nuclear refractive index is strongly correlated with increased DNA content. We therefore propose that an abundance of cells undergoing DNA replication and mitosis may explain the increase in nuclear refractive index observed in both malignant and histologically normal cells from cancer patients. Our findings suggest that nuclear refractive index may be a novel physical parameter for early cancer detection and risk stratification.

A dynamic light scattering technique is implemented using optical coherence tomography (OCT) to measure the change in intracellular motion as cells undergo apoptosis. Acute myeloid leukemia cells were treated with cisplatin and imaged at a frame rate of 166 Hz using a 1300 nm swept-source OCT system at various times over a period of 48 h. Time correlation analysis of the speckle intensities indicated a significant increase in intracellular motion 24 h after treatment. This rise in intracellular motion correlated with histological findings of irregularly shaped and fragmented cells indicative of cell membrane blebbing and fragmentation.

An introduction to the special section by the guest editors.

As an extension of frequency-domain photothermal radiometry, a novel dental-imaging modality, thermophotonic lock-in imaging (TPLI), is introduced. This methodology uses photothermal wave principles and is capable of detecting early carious lesions and cracks on occlusal and approximal surfaces as well as early caries induced by artificial demineralizing solutions. The increased light scattering and absorption within early carious lesions increases the thermal-wave amplitude and shifts the thermal-wave centroid, producing contrast between the carious lesion and the intact enamel in both amplitude and phase images. Samples with artificial and natural occlusal and approximal caries were examined in this study. Thermophotonic effective detection depth is controlled by the modulation frequency according to the well-known concept of thermal diffusion length. TPLI phase images are emissivity normalized and therefore insensitive to the presence of stains. Amplitude images, on the other hand, provide integrated information from deeper enamel regions. It is concluded that the results of our noninvasive, noncontacting imaging methodology exhibit higher sensitivity to very early demineralization than dental radiographs and are in agreement with the destructive transverse microradiography mineral density profiles.

The holmium:YAG (Ho:YAG) laser lithotriptor is capable of operating at high pulse energies, but efficient operation is limited to low pulse rates (∼10 Hz) during lithotripsy. On the contrary, the thulium fiber laser (TFL) is limited to low pulse energies, but can operate efficiently at high pulse rates (up to 1000 Hz). This study compares stone ablation threshold, ablation rate, and retropulsion for the two different Ho:YAG and TFL operation modes. The TFL (λ = 1908 nm) was operated with pulse energies of 5 to 35 mJ, 500-μs pulse duration, and pulse rates of 10 to 400 Hz. The Ho:YAG laser (λ = 2120 nm) was operated with pulse energies of 30 to 550 mJ, 350-μs pulse duration, and a pulse rate of 10 Hz. Laser energy was delivered through 200- and 270-μm-core optical fibers in contact mode with human calcium oxalate monohydrate (COM) stones for ablation studies and plaster-of-Paris stone phantoms for retropulsion studies. The COM stone ablation threshold for Ho:YAG and TFL measured 82.6 and 20.8 J/cm2, respectively. Stone retropulsion with the Ho:YAG laser linearly increased with pulse energy. Retropulsion with TFL was minimal at pulse rates less than 150 Hz, then rapidly increased at higher pulse rates. For minimal stone retropulsion, Ho:YAG operation at pulse energies less than 175 mJ at 10 Hz and TFL operation at 35 mJ at 100 Hz is recommended, with both lasers producing comparable ablation rates. Further development of a TFL operating with both high pulse energies of 100 to 200 mJ and high pulse rates of 100 to 150 Hz may also provide an alternative to the Ho:YAG laser for higher ablation rates, when retropulsion is not a primary concern.

The laser ablation rate of subgingival dental calculus irradiated at a 400-nm-wavelength, 7.4-mJ pulse energy, and 85- and 20-deg irradiation angles is measured using laser triangulation. Three-dimensional images taken before and after irradiation create a removal map with 6-μm axial resolution. Fifteen human teeth with subgingival calculus are irradiated in vitro under a cooling water spray with an ∼300-μm-diam, tenth-order super-Gaussian beam. The average subgingival calculus removal rates for irradiation at 85 and 20 deg are 11.1±3.6 and 11.5±5.9 μm/pulse, respectively, for depth removal and 4.5±1.7×105 and 4.8±2.3×105 μm3/pulse, respectively, for volume removal. The ablation rate is constant at each irradiation site but varies between sites because of the large differences in the physical and optical properties of calculus. Comparison of the average depth- and volume-removal rates does not reveal any dependence on the irradiation angle and is likely due to the surface topology of subgingival calculus samples that overshadows any expected angular dependence.

The aim of this study was to test the hypothesis that in a short-term clinical pilot trial short-pulsed 9.6 μm CO₂-laser irradiation significantly inhibits demineralization in vivo. Twenty-four subjects scheduled for extraction of bicuspids for orthodontic reasons (age 14.9 ± 2.2 years) were recruited. Orthodontic brackets were placed on bicuspids (Transbond XT, 3M). An area next to the bracket was irradiated with a CO₂-laser (Pulse System Inc, Los Alamos, New Mexico), wavelength 9.6 μm, pulse duration 20 μs, pulse repetition rate 20 Hz, beam diameter 1100 μm, average fluence 4.1 ± 0.3J/cm², 20 laser pulses per spot. An adjacent nonirradiated area served as control. Bicuspids were extracted after four and twelve weeks, respectively, for a quantitative assessment of demineralization by cross-sectional microhardness testing. For the 4-week arm the mean relative mineral loss ΔZ (vol% × μm) for the laser treated enamel was 402 ± 85 (mean ± SE), while the control showed significantly higher mineral loss (ΔZ 738 ± 131; P = 0.04, t-test). The difference was even larger after twelve weeks (laser arm ΔZ 135 ± 98; control 1067 ± 254; P = 0.002). The laser treatment produced 46% demineralization inhibition for the 4-week and a marked 87% inhibition for the 12-week arm. This study shows, for the first time in vivo, that the short-pulsed 9.6 μm CO₂-laser irradiation successfully inhibits demineralization of tooth enamel in humans.

Photothermal radiometry and modulated luminescence (PTR-LUM) is an emerging nondestructive methodology applied toward the characterization and quantification of dental caries. We evaluate the efficacy of PTR-LUM in vitro to detect, monitor, and quantify human enamel caries. Artificial caries are created in extracted human molars (n = 15) using an acidified gel system (pH 4.5) for 10 or 40 days. PTR-LUM frequency scans (1 Hz-1 kHz) are performed before and during demineralization. Transverse microradiography (TMR) analysis, the current gold standard, follows at treatment conclusion to determine the mineral loss and depth of the artificially demineralized lesions. A theoretical model is applied to PTR experimental data to evaluate the changes in optothermophysical properties of demineralized enamel as a function of time. Higher optical scattering coefficients and poorer thermophysical properties are characteristic of the growing demineralized lesions, as verified by TMR, where the generated microporosities of the subsurface lesion confine the thermal-wave centroid. Enhanced optical scattering coefficients of demineralized lesions result in poorer luminescence yield due to scattering of both incident and converted luminescent photons. PTR-LUM sensitivity to changes in tooth mineralization coupled with opto-thermophysical property extraction illustrates the technique's potential for nondestructive quantification of enamel caries.

The ability to probe fresh tissue is a key feature to biomedical Raman spectroscopy. However, it is unclear how Raman spectra of calcified tissues are affected by freezing. In this study, six transverse sections of femoral cortical bone were subjected to multiple freeze/thaw cycles and probed using a custom Raman microscope. Significant decreases were observed in the amide I and amide III bands starting after two freeze thaw cycles. Raman band intensities arising from proline residues of frozen tissue appeared consistent with fresh tissue after four cycles. Crystallinity values of bone mineral diminished slightly with freezing and were noticeable after only one freezing. Mineral carbonate levels did not deviate significantly with freezing and thawing. The authors recommend freezing and thawing bone tissue only once to maintain accurate results.

This study aimed to investigate whether swept source optical coherence tomography (SS-OCT) could estimate the lesion depth and mineral loss quantitatively without the use of polarization sensitivity, and to examine a relationship between OCT data and transverse microradiography (TMR) lesion parameters. Twenty-four bovine root dentin specimens were allocated to three groups of 4-, 7-, and 14-day demineralization. Cross-sectional images of the specimens before and after the demineralization were captured by OCT at 1319 nm center wavelength. Following the demineralization, these specimens were cut into sections for TMR analysis. Correlations between the OCT data and TMR lesion parameters were examined. TMR images of the specimens showed cavitated lesions (lesion depth or LDTMR: 200 to 500 μm, ΔZ or mineral loss: 10,000 to 30,000 vol % μm). The OCT images showed "boundaries," suggesting the lesion front. Integrated dB values before and after the demineralization and their difference (RD, RS, and ΔR, respectively) were calculated from the lesion surface to the corrected depth of boundary (LDOCT). A statistically significant correlation was found between LDOCT and LDTMR (p < 0.05, r = 0.68). Similarly, statistically significant correlations were found between ΔZ and RD or ΔR. The OCT showed a potential for quantitative estimation of lesion depth and mineral loss with cavitated dentin lesions in vitro.

We characterized and imaged dental calculus using swept-source optical coherence tomography (SS-OCT). The refractive indices of enamel, dentin, cementum, and calculus were measured as 1.625 ± 0.024, 1.534 ± 0.029, 1.570 ± 0.021, and 2.097 ± 0.094, respectively. Dental calculus leads strong scattering properties, and thus, the region can be identified from enamel with SS-OCT imaging. An extracted human tooth with calculus is covered with gingiva tissue as an in vitro sample for tomographic imaging.

CO2 lasers can be operated at high laser pulse repetition rates for the rapid and precise removal of dental decay. Excessive heat accumulation and peripheral thermal damage is a concern when using high pulse repetition rates. Peripheral thermal damage can adversely impact the mechanical strength of the irradiated tissue, particularly for dentin, and reduce the adhesion characteristics of the modified surfaces. The interpulpal temperature rise was recorded using microthermocouples situated at the roof of the pulp chamber on teeth that were occlusally ablated using a rapidly-scanned CO2 laser operating at 9.3 μm with a pulse duration of 10 to 15 μs and repetition rate of 300 Hz over a 2 min time course. The adhesion strength of laser treated enamel and dentin surfaces was measured for various laser scanning parameters with and without post-ablation acid etching using the single-plane shear test. The mechanical strength of laser-ablated dentin surfaces were determined via the four-point bend test and compared to control samples prepared with 320 grit wet sand paper to simulate conventional preparations. Thermocouple measurements indicated that the temperature remained below ambient temperature if water-cooling was used. There was no discoloration of either dentin or enamel laser treated surfaces, the surfaces were uniformly ablated, and there were no cracks visible. Four-point bend tests yielded mean mechanical strengths of 18.2 N (s.d. = 4.6) for ablated dentin and 18.1 N (s.d. = 2.7) for control (p > 0.05). Shear tests yielded mean bond strengths approaching 30 MPa for both enamel and dentin under certain irradiation conditions. These values were slightly lower than nonirradiated acid-etched control samples. Additional studies are needed to determine if the slightly lower bond strength than the acid-etched control samples is clinically significant.

Autofluorescence spectra were recorded in vitro from dentin, enamel, and whole teeth. The spectra exhibited a broad peak shifted by about 50 to 75 nm from the excitation wavelength and the shape of the spectra remained similar regardless of the excitation wavelength. The maximum of the autofluorescence spectra also exhibited a red-shift that depended upon the laser excitation wavelength. The amplitude of the red-shifted fluorescence spectra produced by 444 and 532 nm excitation lasers were compared to that produced by a 405 nm excitation laser. It was determined that the autofluorescence amplitude was not proportional to the inverse fourth power of the excitation laser wavelength. Therefore, the red-shifted fluorescence is not compatible with the previously proposed mechanism of Raman scattering. Instead, the mechanism giving rise to the laser-induced dental autofluorescence is explained by the red-edge-excitation effect.

A theoretical model that deals with SHG from crystallized type I collagen fiber formed by a bundle of fibrils is established. By introducing a density distribution function of dipoles within fibrils assembly into the dipole theory and combining with structural order (m,l) parameters revealed by quasi-phase-matching (QPM) theory, our established theoretical model comprehensively characterizes both biophysical features of collagen dipoles and the crystalline characteristics of collagen fiber. This new model quantitatively reveals the 3-D distribution of second-harmonic generation (SHG) emission angle (θ,φ) in accordance with the emission power. Results show that fibrils diameter d1 and structural order m, which describes the structural characteristics of collagen fiber along the incident light propagation direction has significant influence on backward/forward SHG emission. The decrease of fibrils diameter d1 induces an increase of the peak SHG emission angle θmax. As d1 decreases to a threshold value, in our case it is around d1 = 150 nm when (m,l) = (1,0), θmax > 90 deg, indicating that backward SHG emission appears. The SHG may have two symmetrical emission distribution lobes or may have only one or two unsymmetrical emission lobes with unequal emission power, depending on the functional area of (m,l) on d1.

A popular phantom in photoacoustic imaging is poly(vinyl alcohol) (PVA) hydrogel fabricated by freezing and thawing (F-T) aqueous solutions of PVA. The material possesses acoustic and optical properties similar to those of tissue. Earlier work characterized PVA gels in small test specimens where temperature distributions during F-T are relatively homogeneous. In this work, in breast-sized samples we observed substantial temperature differences between the shallow regions and the interior during the F-T procedure. We investigated whether spatial variations were also present in the acoustic and optical properties. The speed of sound, acoustic attenuation, and optical reduced scattering coefficients were measured on specimens sampled at various locations in a large phantom. In general, the properties matched values quoted for breast tissue. But while acoustic properties were relatively homogeneous, the reduced scattering was substantially different at the surface compared with the interior. We correlated these variations with gel microstructure inspected using scanning electron microscopy. Interestingly, the phantom's reduced scattering spatial distribution matches the optical properties of the standard two-layer breast model used in x ray dosimetry. We conclude that large PVA samples prepared using the standard recipe make excellent breast tissue phantoms.

As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.

Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm2), infrared laser (780 nm, 40 mW, 1 W/cm2), and red LED (637 ± 15 nm, 40 mW, 1 W/cm2). All applications were punctual and performed with a spot with 0.4 mm2 of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p ≤ 0.05) were used for statistical analysis. After 72 h, phototherapy with low-intensity laser and LED showed no toxicity at the cellular level. It even stimulated methylthiazol tetrazolium assay (MTT) conversion and neutral red uptake of fibroblasts cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p < 0.001 for neutral red uptake). Considering the parameters and protocol of phototherapy used, it can be concluded that phototherapy stimulated the viability of fibroblasts cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced.

We report a hyperspectral imaging system to measure the reflectance spectra of real human irises with high spatial resolution. A set of ocular prosthesis was used as the control condition. Reflectance data were decorrelated by the principal-component analysis. The main conclusion is that spectral complexity of the human iris is considerable: between 9 and 11 principal components are necessary to account for 99% of the cumulative variance in human irises. Correcting image misalignments associated with spontaneous ocular movements did not influence this result. The data also suggests a correlation between the first principal component and different levels of melanin present in the irises. It was also found that although the spectral characteristics of the first five principal components were not affected by the radial and angular position of the selected iridal areas, they affect the higher-order ones, suggesting a possible influence of the iris texture. The results show that hyperspectral imaging in the iris, together with adequate spectroscopic analyses provide more information than conventional colorimetric methods, making hyperspectral imaging suitable for the characterization of melanin and the noninvasive diagnosis of ocular diseases and iris color.

Advances in the functional imaging of cortical hemodynamics have greatly facilitated the understanding of neurovascular coupling. In this study, label-free optical-resolution photoacoustic microscopy (OR-PAM) was used to monitor microvascular responses to direct electrical stimulations of the mouse somatosensory cortex through a cranial opening. The responses appeared in two forms: vasoconstriction and vasodilatation. The transition between these two forms of response was observed in single vessels by varying the stimulation intensity. Marked correlation was found between the current-dependent responses of two daughter vessels bifurcating from the same parent vessel. Statistical analysis of twenty-seven vessels from three different animals further characterized the spatial-temporal features and the current dependence of the microvascular response. Our results demonstrate that OR-PAM is a valuable tool to study neurovascular coupling at the microscopic level.

Almost all diseases, especially cancer and diabetes, manifest abnormal oxygen metabolism. Accurately measuring the metabolic rate of oxygen (MRO2) can be helpful for fundamental pathophysiological studies, and even early diagnosis and treatment of disease. Current techniques either lack high resolution or rely on exogenous contrast. Here, we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution to noninvasively quantify MRO2 in vivo in absolute units. mPAM is the unique modality for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed. Hyperthermia, cryotherapy, melanoma, and glioblastoma were longitudinally imaged in vivo. Counterintuitively, increased MRO2 does not necessarily cause hypoxia or increase oxygen extraction. In fact, early-stage cancer was found to be hyperoxic despite hypermetabolism.

We developed a novel system for imaging and qualitatively analyzing the surface vessels using near-infrared (NIR) radiation using tuned aperture computed tomography (TACT®). The system consisted of a NIR-sensitive CCD camera surrounded by sixty light emitting diodes (with wavelengths alternating between 700 or 810 nm). This system produced thin NIR tomograms, under 0.5 mm in slice thickness. The venous oxygenation index reflecting oxygen saturation levels calculated from NIR tomograms was more sensitive than that from the NIR images. This novel system makes it possible to noninvasively obtain NIR tomograms and accurately analyze changes in oxygen

A photoacoustic tomography system (PAT) using virtual point ultrasonic transducers was developed and applied to image a monkey brain. The custom-built transducers provide a 10-fold greater field-of-view (FOV) than finite-aperture unfocused transducers as well as an improved signal-to-noise ratio (SNR) and reduced artifacts rather than negative-lens transducers. Their tangential resolution, radial resolution, and (SNR) improvements were quantified using tissue phantoms. Our PAT system can achieve high uniformity in both resolution (<1 mm) and SNR (>8) within a large FOV of 6 cm in diameter, even when the imaging objects are enclosed by a monkey skull. The cerebral cortex of a monkey brain was accurately mapped transcranially, through a skull ranging from 2 to 4 mm in thickness. This study demonstrates that PAT can overcome the optical and ultrasound attenuation of a relatively thick skull and can potentially be applied to human neonatal brain imaging.

Optoelectronics techniques to induce protoporphyrin IX fluorescence with topically applied 5-aminolevulinic acid on the oral mucosa have been developed to noninvasively detect oral cancer. Fluorescence imaging enables wide-area screening for oral premalignancy, but the lack of an adequate fluorescence enhancement method restricts the clinical imaging application of these techniques. This study aimed to develop a reliable fluorescence enhancement method to improve PpIX fluorescence imaging systems for oral cancer detection. Three contrast features, red-green-blue reflectance difference, R/B ratio, and R/G ratio, were developed first based on the optical properties of the fluorescence images. A comparative study was then carried out with one negative control and four biopsy confirmed clinical cases to validate the optimal image processing method for the detection of the distribution of malignancy. The results showed the superiority of the R/G ratio in terms of yielding a better contrast between normal and neoplastic tissue, and this method was less prone to errors in detection. Quantitative comparison with the clinical diagnoses in the four neoplastic cases showed that the regions of premalignancy obtained using the proposed method accorded with the expert's determination, suggesting the potential clinical application of this method for the detection of oral cancer.

Early detection of cancer remains the best way to ensure patient survival and quality of life. Squamous cell carcinoma is usually preceded by dysplasia presenting as white, red, or mixed red and white epithelial lesions on the oral mucosa (leukoplakia, erythroplakia). Dysplastic lesions in the form of erythroplakia can carry a risk for malignant conversion of 90%. A noninvasive diagnostic modality would enable monitoring of these lesions at regular intervals and detection of treatment needs at a very early, relatively harmless stage. The specific aim of this work was to test a multimodality approach [three-dimensional optical coherence tomography (OCT) and polarimetry] to noninvasive diagnosis of oral premalignancy and malignancy using the hamster cheek pouch model (nine hamsters). The results were compared to tissue histopathology. During carcinogenesis, epithelial down grow, eventual loss of basement membrane integrity, and subepithelial invasion were clearly visible with OCT. Polarimetry techniques identified a four to five times increased retardance in sites with squamous cell carcinoma, and two to three times greater retardance in dysplastic sites than in normal tissues. These techniques were particularly useful for mapping areas of field cancerization with multiple lesions, as well as lesion margins.

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.

Dynamic analyses of vibrational motion in cell membranes provide a lot of information on the complex dynamic motilities of a red blood cell (RBC). Here, we present the correlation properties of membrane fluctuation in discocyte and spherocyte RBCs by using quantitative phase microscopy (QPM). Since QPM can provide nanometer sensitivity in thickness measurement within a millisecond time scale, we were able to observe the membrane flicking of an RBC in nanometer resolution up to the bandwidth of 50 Hz. The correlation properties of the vibrational motion were analyzed with the detrended fluctuation analysis (DFA) method. Fractal scaling exponent α in the DFA method was calculated for the vibrational motion of a cell surface at various surface points for normal discocyte and abnormal spherocyte RBCs. Measured α values for normal RBCs are distributed between 0.7 and 1.0, whereas those for abnormal spherocyte RBCs are within a range from 0.85 to 1.2. We have also verified that the vibrational motion of background fluid outside of a cell has an α value close to 0.5, which is a typical property of an uncorrelated white noise.

A Fourier domain optical coherence tomography setup is presented built around an optical configuration that exhibits Talbot bands. A low astigmatism spectrometer is used, employing a spherical mirror and a cylindrical lens between a diffraction grating and a linear CCD camera. To produce Talbot bands, the two interferometer beams-object and reference-are laterally shifted in respect to each other in their way toward the diffraction grating. This allows attenuation of mirror terms and optimization of the sensitivity profile. We evaluate the optimization of the sensitivity profile with depth, in respect to its overall strength and its position peak, which can be shifted toward a larger optical path difference in the interferometer. We demonstrate the efficiency of such a configuration at large depths by imaging a thick phantom and human skin in vivo for different values of the lateral distance between the two beams.

The location and ischemia extent are two important parameters for evaluating the acute myocardial ischemia (AMI). A focused-transducer-based photoacoustic imaging method was employed to assess time-dependent AMI. Our preliminary results show that the photoacoustic signal could identify the myocardium. The intensity and area of photoacoustic images of myocardium could be used for characterizing the ischemia extent and scope of myocardial ischemia. The results also imply that the intensity and area of photoacoustic images are the rapid fall of an exponential model with an increase of delaying time after the left anterior descending coronary artery (LAD) occlusion. These experimental results were consistent with the clinical characteristics. The findings suggest that the photoacoustic imaging be a potential tool for the real-time assessment of acute myocardial ischemia during surgical operation.

Dry eye syndrome is one of the most commonly reported eye health conditions. Dynamic-area high-speed videokeratoscopy (DA-HSV) represents a promising alternative to the most invasive clinical methods for the assessment of the tear film surface quality (TFSQ), particularly as Placido-disk videokeratoscopy is both relatively inexpensive and widely used for corneal topography assessment. Hence, improving this technique to diagnose dry eye is of clinical significance and the aim of this work. First, a novel ray-tracing model is proposed that simulates the formation of a Placido image. This model shows the relationship between tear film topography changes and the obtained Placido image and serves as a benchmark for the assessment of indicators of the ring's regularity. Further, a novel block-feature TFSQ indicator is proposed for detecting dry eye from a series of DA-HSV measurements. The results of the new indicator evaluated on data from a retrospective clinical study, which contains 22 normal and 12 dry eyes, have shown a substantial improvement of the proposed technique to discriminate dry eye from normal tear film subjects. The best discrimination was obtained under suppressed blinking conditions. In conclusion, this work highlights the potential of the DA-HSV as a clinical tool to diagnose dry eye syndrome.

Three-dimensional optical coherence tomography (3D-OCT) was used to image the structure and pathology of skeletal muscle tissue from the treadmill-exercised mdx mouse model of human Duchenne muscular dystrophy. Optical coherence tomography (OCT) images of excised muscle samples were compared with co-registered hematoxylin and eosin-stained and Evans blue dye fluorescence histology. We show, for the first time, structural 3D-OCT images of skeletal muscle dystropathology well correlated with co-located histology. OCT could identify morphological features of interest and necrotic lesions within the muscle tissue samples based on intrinsic optical contrast. These findings demonstrate the utility of 3D-OCT for the evaluation of small-animal skeletal muscle morphology and pathology, particularly for studies of mouse models of muscular dystrophy.

Diffuse optical tomography has shown promising results as a tool for breast cancer screening and monitoring response to chemotherapy. Dynamic imaging of the transient response of the breast to an external stimulus, such as pressure or a respiratory maneuver, can provide additional information that can be used to detect tumors. We present a new digital continuous-wave optical tomography system designed to simultaneously image both breasts at fast frame rates and with a large number of sources and detectors. The system uses a master-slave digital signal processor-based detection architecture to achieve a dynamic range of 160 dB and a frame rate of 1.7 Hz with 32 sources, 64 detectors, and 4 wavelengths per breast. Included is a preliminary study of one healthy patient and two breast cancer patients showing the ability to identify an invasive carcinoma based on the hemodynamic response to a breath hold.

Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.

The formation of protein complexes or clusters in the plasma membrane is essential for many biological processes, such as signaling. We develop a tool, based on single-molecule microscopy, for following cluster formation in vivo. Detection and tracing of single autofluorescent proteins have become standard biophysical techniques. The determination of the number of proteins in a cluster, however, remains challenging. The reasons are (i) the poor photophysical stability and complex photophysics of fluorescent proteins and (ii) noise and autofluorescent background in live cell recordings. We show that, despite those obstacles, the accurate fraction of signals in which a certain (or set) number of labeled proteins reside, can be determined in an accurate an robust way in vivo. We define experimental conditions under which fluorescent proteins exhibit predictable distributions of intensity and quantify the influence of noise. Finally, we confirm our theoretical predictions by measurements of the intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in living cells. Quantification of the average number of EYFP-C10HRAS chimeras in diffraction-limited spots finally confirm that the membrane anchor of human Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of living Chinese hamster ovary cells.

To measure the retardation distribution of the optic retinal nerve fiber layer (RNFL) from a single image, we have developed a new polarization analysis system that is able to detect the Stokes vector using a fundus camera. The polarization analysis system is constructed with a CCD area image sensor, a linear polarizing plate, a microphase plate array, and a circularly polarized light illumination unit. In this system, the Stokes vector expressing the whole state of polarization is detected, and the influence of the background scattering in the retina and of the retardation caused by the cornea are numerically eliminated. The measurement method is based on the hypothesis that the retardation process of the eye optics can be quantified by a numerical equation that consists of a retardation matrix of all the polarization components. We show the method and the measurement results for normal eyes. Our results indicate that the present method may provide a useful means for the evaluation of retardation distribution of the RNFL.

Clustered small breast lesions may be present in the neighboring areas and are difficult to accurately resolve and quantify in diffuse optical tomography. In addition, larger cancers are often accompanied by clustered satellite lesions in the neighboring areas, which are also difficult to resolve and quantify. To improve the light quantification of clustered lesions, a new multi-zone reconstruction algorithm guided by co-registered ultrasound (US) was investigated using simulations, phantoms, and clinical examples. This method separated one larger region-of-interest (ROI) into several ROIs based on the location information provided by co-registered US. In general, the single-ROI method cannot resolve two smaller targets when their separations were less than 2.5 cm and the depth was greater than 2.0 cm. The multi-zone reconstruction method improved the resolving ability and reconstruction accuracy. As a result, two targets located at 2.5 cm depth with separation greater than 2.0 cm could be distinguished, and reconstruction improved by more than 20% as compared with that of the single-ROI method. When two targets, one larger and one smaller, were located closer to each other, the location of the reconstructed absorption mass was shifted toward the larger target and the quantification of the smaller target was limited.

We describe the implementation and use of an adaptive optics loop in the imaging path of a commercial wide field microscope. We show that it is possible to maintain the optical performances of the original microscope when imaging through aberrant biological samples. The sources used for illuminating the adaptive optics loop are spectrally independent, in excitation and emission, from the sample, so they do not appear in the final image, and their use does not contribute to the sample bleaching. Results are compared with equivalent images obtained with an identical microscope devoid of adaptive optics system.

A relative humidity (RH) sensor based on a hybrid fiber grating coated with polyvinyl alcohol is proposed and demonstrated experimentally. The hybrid fiber grating is formed by superimposing a normal fiber Bragg grating (FBG) and a tilted-FBG around the same position of a single-mode fiber so that it can work in the reflection mode. Optical power of the reflected signal changes with the refractive index of the moisture sensitive polyvinyl alcohol, and humidity measurement can be realized. Experimental results show that the measurement range is 30 to 95% with the maximum sensitivity of 0.737 nW/% RH. The average response time is ∼2 s and the measurement is nearly insensitive to temperature. Compared with the wavelength detection method used in normal FBG-based relative humidity sensors, the intensity demodulation method in this report is simpler and more cost-efficient.

We have demonstrated that spatially modulated quantitative spectroscopy (SMoQS) is capable of extracting absolute optical properties from homogeneous tissue simulating phantoms that span both the visible and near-infrared wavelength regimes. However, biological tissue, such as skin, is highly structured, presenting challenges to quantitative spectroscopic techniques based on homogeneous models. In order to more accurately address the challenges associated with skin, we present a method for depth-resolved optical property quantitation based on a two layer model. Layered Monte Carlo simulations and layered tissue simulating phantoms are used to determine the efficacy and accuracy of SMoQS to quantify layer specific optical properties of layered media. Initial results from both the simulation and experiment show that this empirical method is capable of determining top layer thickness within tens of microns across a physiological range for skin. Layer specific chromophore concentration can be determined to <±10% the actual values, on average, whereas bulk quantitation in either visible or near infrared spectroscopic regimes significantly underestimates the layer specific chromophore concentration and can be confounded by top layer thickness.

Interstitial fiber-optic-based approaches used in both diagnostic and therapeutic applications rely on localized light-tissue interactions. We present an optical technique to identify spectrally and spatially specific exogenous chromophores in highly scattering turbid media. Point radiance spectroscopy is based on directional light collection at a single point with a side-firing fiber that can be rotated up to 360 deg. A side firing fiber accepts light within a well-defined, solid angle, thus potentially providing an improved spatial resolution. Measurements were performed using an 800-μm diameter isotropic spherical diffuser coupled to a halogen light source and a 600 μm, ∼43 deg cleaved fiber (i.e., radiance detector). The background liquid-based scattering phantom was fabricated using 1% Intralipid. Light was collected with 1 deg increments through 360 deg-segment. Gold nanoparticles , placed into a 3.5-mm diameter capillary tube were used as localized scatterers and absorbers introduced into the liquid phantom both on- and off-axis between source and detector. The localized optical inhomogeneity was detectable as an angular-resolved variation in the radiance polar plots. This technique is being investigated as a potential noninvasive optical modality for prostate cancer monitoring.

The effectiveness of peripheral nerve blocks is highly dependent on the accuracy at which the needle tip is navigated to the target injection site. Even when electrical stimulation is utilized in combination with ultrasound guidance, determining the proximity of the needle tip to the target region close to the nerve can be challenging. Optical reflectance spectroscopy could provide additional information about tissues that is complementary to these navigation methods. We demonstrate a novel needle stylet for acquiring spectra from tissue at the tip of a commercial 20-gauge needle. The stylet has integrated optical fibers that deliver broadband light to tissue and receive scattered light. Two spectrometers resolve the light that is received from tissue across the wavelength range of 500-1600 nm. In our pilot study, measurements are acquired from a postmortem dissection of the brachial plexus of a swine. Clear differences are observed between spectra acquired from nerves and those acquired from adjacent tissue structures. We conclude that spectra acquired with the stylet have the potential to increase the accuracy with which peripheral nerve blocks are performed.

A prototype fiber-optic reflectance-mode pulse oximetry sensor and measurement system is developed for the purposes of estimating arterial oxygen saturation in the esophagus. A dedicated probe containing miniature right-angled glass prisms coupled to light sources and a photodetector by means of optical fibers is designed and used to record photoplethysmographic (PPG) signals from the esophageal epithelium in anesthetized patients. The probe is inserted simply by an anesthesiologist in all cases, and signals are recorded successfully in all but one of 20 subjects, demonstrating that esophageal PPG signals can be reliably obtained. The mean value of the oxygen saturation recorded from the esophagus for all subjects is 94.0 ± 4.0%. These results demonstrate that SpO2 may be estimated in the esophagus using a fiber-optic probe.

The risk of local recurrence for breast cancers is strongly correlated with the presence of a tumor within 1 to 2 mm of the surgical margin on the excised specimen. Previous experimental and theoretical results suggest that spatially offset Raman spectroscopy (SORS) holds much promise for intraoperative margin analysis. Based on simulation predictions for signal-to-noise ratio differences among varying spatial offsets, a SORS probe with multiple source-detector offsets was designed and tested. It was then employed to acquire spectra from 35 frozen-thawed breast tissue samples in vitro. Spectra from each detector ring were averaged to create a composite spectrum with biochemical information covering the entire range from the tissue surface to ∼2 mm below the surface, and a probabilistic classification scheme was used to classify these composite spectra as "negative" or "positive" margins. This discrimination was performed with 95% sensitivity and 100% specificity, or with 100% positive predictive value and 94% negative predictive value.

Optical techniques toward the realization of sensitive and selective biosensing platforms have received considerable attention in recent times. Techniques based on interferometry, surface plasmon resonance, and waveguides have all proved popular, while spectroscopy in particular offers much potential. Raman spectroscopy is an information-rich technique in which the vibrational frequencies reveal much about the structure of a compound, but it is a weak process and offers poor sensitivity. In response to this problem, surface-enhanced Raman scattering (SERS) has received much attention, due to significant increases in sensitivity instigated by bringing the sample into contact with an enhancing substrate. Here we discuss a facile and rapid technique for the detection of pterins using SERS-active colloidal silver suspensions. Pterins are a family of biological compounds that are employed in nature in color pigmentation and as facilitators in metabolic pathways. In this work, small volumes of xanthopterin, isoxanthopterin, and 7,8-dihydrobiopterin have been examined while adsorbed to silver colloids. Limits of detection have been examined for both xanthopterin and isoxanthopterin using a 10-s exposure to a 12 mW 532 nm laser, which, while showing a trade-off between scan time and signal intensity, still provides the opportunity for the investigation of simultaneous detection of both pterins in solution.

Light scattering in the normally white sclera prevents diagnostic imaging or delivery of a focused laser beam to a target in the underlying choroid layer. In this study, we examine optical clearing of the sclera and changes in blood flow resulting from the application of glycerol to the sclera of rabbits. Recovery dynamics are monitored after the application of saline. The speed of clearing for injection delivery is compared to the direct application of glycerol through an incision in the conjunctiva. Although, the same volume of glycerol was applied, the sclera cleared much faster (5 to 10 s) with the topical application of glycerol compared to the injection method (3 min). In addition, the direct topical application of glycerol spreads over a larger area in the sclera than the latter method. A diffuse optical spectroscopy system provided spectral analysis of the remitted light every two minutes during clearing and rehydration. Comparison of measurements to those obtained from phantoms with various absorption and scattering properties provided estimates of the absorption coefficient and reduced scattering coefficient of rabbit eye tissue.

Raman spectroscopy was performed on optically trapped red blood cells (RBCs) from blood samples of healthy volunteers (h-RBCs) and from patients suffering from P. vivax infection (m-RBCs). A significant fraction of m-RBCs produced Raman spectra with altered characteristics relative to h-RBCs. The observed spectral changes suggest a reduced oxygen-affinity or right shifting of the oxygen-dissociation curve for the intracellular hemoglobin in a significant fraction of m-RBCs with respect to its normal functional state.

With the advance of computer and photonics technology, imaging photoplethysmography [(PPG), iPPG] can provide comfortable and comprehensive assessment over a wide range of anatomical locations. However, motion artifact is a major drawback in current iPPG systems, particularly in the context of clinical assessment. To overcome this issue, a new artifact-reduction method consisting of planar motion compensation and blind source separation is introduced in this study. The performance of the iPPG system was evaluated through the measurement of cardiac pulse in the hand from 12 subjects before and after 5 min of cycling exercise. Also, a 12-min continuous recording protocol consisting of repeated exercises was taken from a single volunteer. The physiological parameters (i.e., heart rate, respiration rate), derived from the images captured by the iPPG system, exhibit functional characteristics comparable to conventional contact PPG sensors. Continuous recordings from the iPPG system reveal that heart and respiration rates can be successfully tracked with the artifact reduction method even in high-intensity physical exercise situations. The outcome from this study thereby leads to a new avenue for noncontact sensing of vital signs and remote physiological assessment, with clear applications in triage and sports training.

The brain activity during cooperation as a form of social process is studied. We investigate the relationship between coinstantaneous brain-activation signals of multiple participants and their cooperative-task performance. A wearable near-infrared spectroscopy (NIRS) system is used for simultaneously measuring the brain activities of two participants. Each pair of participants perform a cooperative task, and their relative changes in cerebral blood are measured with the NIRS system. As for the task, the participants are told to count 10 s in their mind after an auditory cue and press a button. They are also told to adjust the timing of their button presses to make them as synchronized as possible. Certain information, namely, the "intertime interval" between the two button presses of each participant pair and which of the participants was the faster, is fed back to the participants by a beep sound after each trial. When the spatiotemporal covariance between the activation patterns of the prefrontal cortices of each participant is higher, the intertime interval between their button-press times was shorter. This result suggests that the synchronized activation patterns of the two participants' brains are associated with their performance when they interact in a cooperative task.

Photoplethysmography is used in various areas such as vital sign measurement, vascular characteristics analysis, and autonomic nervous system assessment. Photoplethysmographic signals are composed of ac and dc, but it is difficult to find research about the interaction of photoplethysmographic components. This study suggested a model equation combining two Lambert-Beer equations at the onset and peak points of photoplethysmography to evaluate ac characteristics, and verified the model equation through simulation and experiment. In the suggested equation, ac was dependent on dc and optical path length. In the simulation, dc was inversely proportionate to ac sensitivity (slope), and ac and optical path length were proportionate. When dc increased from 10% to 90%, stabilized ac decreased from 1 to 0.89 ± 0.21, and when optical path length increased from 10% to 90%, stabilized ac increased from 1 to 1.53 ± 0.40.

We investigate the effects of a novel bioactive material (Biosilicate®) and low-level laser therapy (LLLT), at 60 J/cm2, on bone-fracture consolidation in osteoporotic rats. Forty female Wistar rats are submitted to the ovariectomy, to induce osteopenia. Eight weeks after the ovariectomy, the animals are randomly divided into four groups, with 10 animals each: bone defect control group; bone defect filled with Biosilicate group; bone defect irradiated with laser at 60 J/cm2 group; bone defect filled with Biosilicate and irradiated with LLLT, at 60 J/cm2 group. Laser irradiation is initiated immediately after surgery and performed every 48 h for 14 days. Histopathological analysis points out that bone defects are predominantly filled with the biomaterial in specimens treated with Biosilicate. In the 60-J/cm2 laser plus Biosilicate group, the biomaterial fills all bone defects, which also contained woven bone and granulation tissue. Also, the biomechanical properties are increased in the animals treated with Biosilicate associated to lasertherapy. Our results indicate that laser therapy improves bone repair process in contact with Biosilicate as a result of increasing bone formation as well as indentation biomechanical properties.

Induction of heat shock protein (Hsp) expression correlates with cytoprotection, reduced tissue damage, and accelerated healing in animal models. Since Hsps are transcriptionally activated in response to stress, they can act as stress indicators in burn injury or surgical procedures that produce heat and thermal change. A fast in vivo readout for induction of Hsp transcription in tissues would allow for the study of these proteins as therapeutic effect mediators and reporters of thermal stress/damage. We used a transgenic reporter mouse in which a luciferase expression is controlled by the regulatory region of the inducible 70 kilodalton (kDa) Hsp as a rapid readout of cellular responses to laser-mediated thermal stress/injury in mouse skin. We assessed the pulse duration dependence of the Hsp70 expression after irradiation with a CO2 laser at 10.6 μm in wavelength over a range of 1000 to 1 ms. Hsp70 induction varied with changes in laser pulse durations and radiant exposures, which defined the ranges at which thermal activation of Hsp70 can be used to protect cells from subsequent stress, and reveals the window of thermal stress that tissues can endure.

We report experimental results on the inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by a visible femtosecond laser. Our results suggest that inactivation of virus and bacterium by a visible femtosecond laser involves completely different mechanisms. Inactivation of viruses by a visible femtosecond laser involves the breaking of hydrogen/hydrophobic bonds or the separation of the weak protein links in the protein shell of a viral particle. In contrast, inactivation of bacteria is related to the damage of their DNAs due to irradiation of a visible femtosecond laser. Possible mechanisms for the inactivation of viruses and bacteria are discussed.