We present a new method for the accurate estimation of diffuse reflectance spectra from RGB values based on Wiener estimation. In the proposed method, a system matrix obtained from the original RGB values is combined with a set of synthetic optical filters to generate another three values corresponding to new colors. A modified Wiener matrix can then be created with the original RGB values and the new color values, which will yield a more accurate estimation because of the new color information that has been incorporated. This method was tested on in vivo color measurements from 200 skin sites in 10 volunteers. The results show that the proposed method is able to improve the estimation accuracy significantly compared with the traditional Wiener estimation method. The fast speed of this method may enable the estimation of diffuse reflectance spectra at multiple tissue locations from color images in real time, which provides a cost-effective alternative to spectral imaging with the additional advantage of high spectral resolution.

We developed optical coherence photoacoustic microscopy (OC-PAM) to demonstrate that the functions of optical coherence tomography (OCT) and photoacoustic microscopy (PAM) can be achieved simultaneously by using a single illuminating light source. We used a pulsed broadband laser centered at 580 nm and detected the absorbed photons through photoacoustic detection and the back-scattered photons with an interferometer. In OC-PAM, each laser pulse generates both one OCT A-line and one PAM A-line simultaneously; as a result, the two imaging modalities are intrinsically co-registered in the lateral directions. In vivo images of the mouse ear were acquired to demonstrate the capabilities of OC-PAM.

A method is proposed that utilizes the advantages of optical ultrasound detection in two-dimensional photoacoustic section imaging, combining an optical interferometer with an acoustic mirror. The concave mirror has the shape of an elliptical cylinder and concentrates the acoustic wave generated around one focal line in the other one, where an optical beam probes the temporal evolution of acoustic pressure. This yields line projections of the acoustic sources at distances corresponding to the time of flight, which, after rotating the sample about an axis perpendicular to the optical detector, allows reconstruction of a section using the inverse Radon transform. A resolution of 120 µm within and 1.5 mm between the sections can be obtained with the setup. Compared to a bare optical probe beam, the signal-to-noise ratio (SNR) is seven times higher with the mirror. Furthermore, the imaging system is tested on a biological sample.

We demonstrate theoretically and experimentally that the phase retardance and relative optic-axis orientation of a sample can be calculated without prior knowledge of the actual value of the phase modulation amplitude when using a polarization-sensitive optical coherence tomography system based on continuous polarization modulation (CPM-PS-OCT). We also demonstrate that the sample Jones matrix can be calculated at any values of the phase modulation amplitude in a reasonable range depending on the system effective signal-to-noise ratio. This has fundamental importance for the development of clinical systems by simplifying the polarization modulator drive instrumentation and eliminating its calibration procedure. This was validated on measurements of a three-quarter waveplate and an equine tendon sample by a fiber-based swept-source CPM-PS-OCT system.

Through a combination of a deflective phase-only diffractive spatial light modulator (SLM) and galvo scanners, an addressable multiregional and multifocal multiphoton microscope (AM-MMM) is developed. The SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple beamlet arrays with addressable shapes and sizes that match the regions of interest on the sample. Compared with conventional multifocal multiphoton microscope (MMM), AM-MMM achieves the effective use of the laser power with an increase of imaging rate and a decrease of photodamage without sacrifice of resolution.

We report an experimental investigation of time-reversed ultrasonically encoded optical focusing in biological tissue. This technology combines the concepts of optical phase conjugation and ultrasound modulation of diffused coherent light. The ultrasonically encoded (or tagged) diffused light from a tissue sample is collected in reflection mode and interferes with a reference light in a photorefractive crystal (used as a phase conjugation mirror) to form a hologram. Then a time-reversed copy of the tagged light is generated and traces back the original trajectories to the ultrasonic focus inside the tissue sample. With our current setup, we can achieve a maximum penetration depth of 5 mm in a chicken breast sample and image optical contrasts within a tissue sample with a spatial resolution approximately equaling 1/√2 of the ultrasound focal diameter.

A simple analytic method of estimating the error involved in using an approximate boundary condition for diffuse radiation in two adjoining scattering media with differing refractive indices is presented. The method is based on asymptotic planar fluences and enables the relative error to be readily evaluated without recourse to Monte Carlo simulation. Three examples of its application are considered: (1) evaluating the error in calculating the diffuse fluences at a boundary between two media with differing refractive index and dissimilar scattering properties, (2) the dependence of the relative error in a multilayer medium with discontinuous refractive index on the ratio of the reduced scattering coefficient to the absorption coefficient μs′/μa, and (3) the parametric dependence of the error in the radiant flux Js at the surface of a three-layer medium. The error is significant for strongly forward-biased scattering media with non-negligible absorption and is cumulative in multilayered media with refractive index increments between layers.

The safety of at-home tooth bleaching, based upon carbamide peroxide (CP) or hydrogen peroxide (HP) as the active agent, has been questioned. The aim of the present study was to investigate the effects of three differently concentrated home-applied bleaching agents on human enamel under in situ conditions. Sixty specimens were divided randomly into four groups and treated with 10% CP, 15% CP, 20% CP, and distilled water, respectively. Raman spectroscopy, attenuated total reflectance-infrared (ATR-IR) spectroscopy, atomic force microscopy (AFM), microhardness, and fracture toughness (FT) measurements were conducted to determine variations on enamel structure and mechanical properties before and after the bleaching process. Raman revealed little variation of Raman relative intensity after treatment with CP, which was consistent with the results of ATR-IR, AFM, and microhardness analyses. In addition, laser-induced fluorescence (LIF) intensity, and FT showed significant decreases on CP-treated specimens. These findings suggested there were minimal demineralization effects of the three at-home bleaching agents on enamel in situ. However, the decrease of LIF intensity and FT on enamel seemed to be inevitable.

Polarization interferometry (PI) techniques, which are able to improve surface plasmon resonance (SPR) sensing performance and reduce restrictions on allowable parameters of SPR-supporting metal films, have been experimentally realized only in SPR sensors using monochromatic light as a source. Wavelength-interrogation SPR sensors modulated by PI techniques have not been reported due to the wavelength-sensitive characterization of PI phase compensators. In this work we develop a specially designed rhombic prism for phase compensating which is totally insensitive to wavelength. For the first time we successfully apply PI technique to a wavelength-interrogation SPR imager. This imager is able to offer two-dimensional imaging of the whole array plane. As a result of PI modulation, resolutions of 1.3×10−6 refractive index unit (RIU) under the normal condition and 3.9×10−7 RIU under a more time-consuming condition are acquired. The application of this imager was demonstrated by reading microarrays for identification of bacteria, and SPR results were confirmed by means of fluorescence imaging.

With no sufficient screening test for ovarian cancer, a method to evaluate the ovarian disease state quickly and nondestructively is needed. The authors have applied a wide-field spectral imager to freshly resected ovaries of 30 human patients in a study believed to be the first of its magnitude. Endogenous fluorescence was excited with 365-nm light and imaged in eight emission bands collectively covering the 400- to 640-nm range. Linear discriminant analysis was used to classify all image pixels and generate diagnostic maps of the ovaries. Training the classifier with previously collected single-point autofluorescence measurements of a spectroscopic probe enabled this novel classification. The process by which probe-collected spectra were transformed for comparison with imager spectra is described. Sensitivity of 100% and specificity of 51% were obtained in classifying normal and cancerous ovaries using autofluorescence data alone. Specificity increased to 69% when autofluorescence data were divided by green reflectance data to correct for spatial variation in tissue absorption properties. Benign neoplasm ovaries were also found to classify as nonmalignant using the same algorithm. Although applied ex vivo, the method described here appears useful for quick assessment of cancer presence in the human ovary.

We present a novel method for real-time 3-D body-shape measurement during breathing based on the laser multiple-line triangulation principle. The laser projector illuminates the measured surface with a pattern of 33 equally inclined light planes. Simultaneously, the camera records the distorted light pattern from a different viewpoint. The acquired images are transferred to a personal computer, where the 3-D surface reconstruction, shape analysis, and display are performed in real time. The measured surface displacements are displayed with a color palette, which enables visual feedback to the patient while breathing is being taught. The measuring range is approximately 400×600×500  mm in width, height, and depth, respectively, and the accuracy of the calibrated apparatus is ±0.7  mm. The system was evaluated by means of its capability to distinguish between different breathing patterns. The accuracy of the measured volumes of chest-wall deformation during breathing was verified using standard methods of volume measurements. The results show that the presented 3-D measuring system with visual feedback has great potential as a diagnostic and training assistance tool when monitoring and evaluating the breathing pattern, because it offers a simple and effective method of graphical communication with the patient.

Extracellular skin structures in human skin are impaired during intrinsic and extrinsic aging. Assessment of these dermal changes is conducted by subjective clinical evaluation and histological and molecular analysis. We aimed to develop a new parameter for the noninvasive quantitative determination of dermal skin alterations utilizing the high-resolution three-dimensional multiphoton laser scanning microscopy (MPLSM) technique. To quantify structural differences between chronically sun-exposed and sun-protected human skin, the respective collagen-specific second harmonic generation and the elastin-specific autofluorescence signals were recorded in young and elderly volunteers using the MPLSM technique. After image processing, the elastin-to-collagen ratio (ELCOR) was calculated. Results show that the ELCOR parameter of volar forearm skin significantly increases with age. For elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area is significantly augmented compared to the sun-protected upper arm area. Based on the MPLSM technology, we introduce the ELCOR parameter as a new means to quantify accurately age-associated alterations in the extracellular matrix.

The aim of this study was to evaluate the diagnostic capabilities of a laser fluorescence tool DIAGNOdent (KaVo, Biberach, Germany) and two light-emitting diode fluorescence tools-Spectra Caries Detection Aid (AIR TECHNIQUES, Melville, NY), and SOPROLIFE light-induced fluorescence evaluator in daylight and blue florescence mode (SOPRO, ACTEON Group, La Ciotat, France)-in comparison to the caries detection and assessment system (ICDAS-II) in detection of caries lesions. In 100 subjects (age 23.4±10.6 years), 433 posterior permanent unrestored teeth were examined. On the occlusal surfaces, up to 1066 data points for each assessment method were available for statistical evaluation, including 1034 ICDAS scores (intra-examiner kappa = 0.884). For the SOPROLIFE tool, a new caries-scoring system was developed. Per assessment tool each average score for one given ICDAS code was significantly different from the one for another ICDAS code. Normalized data linear regression revealed that both SOPROLIFE assessment tools allowed for best caries score discrimination followed by DIAGNOdent and Spectra Caries Detection Aid. The area under the receiver operating characteristics curve calculations showed the same grading sequence when cutoff point ICDAS codes 0-1-2 were grouped together. Sensitivity and specificity values at the same cutoff were calculated (DIAGNOdent 87/66, Spectra Caries Detection Aid 93/37, SOPROLIFE 93/63, SOPROLIFE blue fluorescence 95/55.)

The ability to accurately measure velocity within the embryonic zebrafish heart, at high spatial and temporal resolution, enables further insight into the effects of hemodynamics on heart development. Unfortunately, currently available techniques are unable to provide the required resolution, both spatial and temporal, for detailed analysis. Advances in imaging hardware are allowing bright field imaging combined with particle image velocimetry to become a viable technique for the broader community at the required spatial and temporal resolutions. While bright field imaging offers the necessary temporal resolution, this approach introduces heart wall artifacts that interfere with accurate velocity measurement. This study presents a technique for cardiac-phase filtering of bright field images to remove the heart wall and improve velocimetry measurements. Velocity measurements were acquired for zebrafish embryos ranging from 3 to 6 days postfertilization. Removal of the heart wall was seen to correct a severe (3-fold) underestimation in velocity measurements obtained without filtering. Additionally, velocimetry measurements were used to quantitatively detect developmental changes in cardiac performance in vivo, investigating both changes in contractile period and maximum velocities present through the ventricular-bulbar valve.

An essential and indispensable component of automated microscopy framework is the automatic focusing system, which determines the in-focus position of a given field of view by searching the maximum value of a focusing function over a range of z-axis positions. The focus function and its computation time are crucial to the accuracy and efficiency of the system. Sixteen focusing algorithms were analyzed for histological and histopathological images. In terms of accuracy, results have shown an overall high performance by most of the methods. However, we included in the evaluation study other criteria such as computational cost and focusing curve shape which are crucial for real-time applications and were used to highlight the best practices.

The objective of this study was to assess the ability of combined photothermal wave (PTW) imaging and optical coherence tomography (OCT) to detect, and further characterize the distribution of macrophages (having taken up plasmonic gold nanorose as a contrast agent) and lipid deposits in atherosclerotic plaques. Aortas with atherosclerotic plaques were harvested from nine male New Zealand white rabbits divided into nanorose- and saline-injected groups and were imaged by dual-wavelength (800 and 1210 nm) multifrequency (0.1, 1 and 4 Hz) PTW imaging in combination with OCT. Amplitude PTW images suggest that lateral and depth distribution of nanorose-loaded macrophages (confirmed by two-photon luminescence microscopy and RAM-11 macrophage stain) and lipid deposits can be identified at selected modulation frequencies. Radiometric temperature increase and modulation amplitude of superficial nanoroses in response to 4 Hz laser irradiation (800 nm) were significantly higher than native plaque (P<0.001). Amplitude PTW images (4 Hz) were merged into a coregistered OCT image, suggesting that superficial nanorose-loaded macrophages are distributed at shoulders on the upstream side of atherosclerotic plaques (P<0.001) at edges of lipid deposits. Results suggest that combined PTW-OCT imaging can simultaneously reveal plaque structure and composition, permitting characterization of nanorose-loaded macrophages and lipid deposits in atherosclerotic plaques.

We have investigated depth-resolved cellular structures of unmodified fresh human scalp hairs with ultrahigh-resolution full-field optical coherence tomography (FF-OCT). The Linnik-type white light interference microscope has been home-implemented to observe the micro-internal layers of human hairs in their natural environment. In hair shafts, FF-OCT has qualitatively revealed the cellular hair compartments of cuticle and cortex layers involved in keratin filaments and melanin granules. No significant difference between black and white hair shafts was observed except for absence of only the melanin granules in the white hair, reflecting that the density of the melanin granules directly affects the hair color. Anatomical description of plucked hair bulbs was also obtained with the FF-OCT in three-dimensions. We expect this approach will be useful for evaluating cellular alteration of natural hairs on cosmetic assessment or diagnosis of hair diseases.

A prototype instrument developed to provide simultaneously ordinary visual endoscopy together with near infrared (NIR) autofluorescence imaging via parallel image acquisition is demonstrated. The two images are recorded concurrently and the instrument interfaces with any ordinary endoscope. Preliminary results of a pilot study focused on imaging of bladder tumors in vivo using this instrumentation are presented. The experimental results demonstrate the capabilities of this instrumentation design, imaging methodology, and define the current limitation for further development of the system.

The metabolic rate of oxygen consumption, an important indicator of tissue metabolism, can be expressed as the change of net blood oxygen flux into and out of a tissue region per 100 g of tissue. In this work, we propose a photoacoustic and Doppler ultrasound method for imaging local blood oxygen flux of a single vessel. An imaging system for combined photoacoustic and high-frequency ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and Doppler ultrasound images are co-registered. Since the mean flow speed can be measured by color Doppler ultrasound, the vessel cross-sectional area can be measured by power Doppler or structural photoacoustic imaging, and multi-wavelength photoacoustic methods can be used to estimate oxygen saturation (sO₂) and total concentration of haemoglobin (CHb), all of the parameters necessary for oxygen flux estimation can be provided. The accuracy of the flow speed and sO₂ estimation has been investigated. In vitro sheep blood phantom experiments have been performed at different sO₂ levels and mean flow speeds. Blood oxygen flux has been estimated, and the uncertainty of the measurement has been quantified.

Reconstruction algorithms for imaging fluorescence in near infrared ranges usually normalize fluorescence light with respect to excitation light. Using this approach, we investigated the influence of absorption and scattering heterogeneities on quantification accuracy when assuming a homogeneous model and explored possible reconstruction improvements by using a heterogeneous model. To do so, we created several computer-simulated phantoms: a homogeneous slab phantom (P1), slab phantoms including a region with a two- to six-fold increase in scattering (P2) and in absorption (P3), and an atlas-based mouse phantom that modeled different liver and lung scattering (P4). For P1, reconstruction with the wrong optical properties yielded quantification errors that increased almost linearly with the scattering coefficient while they were mostly negligible regarding the absorption coefficient. This observation agreed with the theoretical results. Taking the quantification of a homogeneous phantom as a reference, relative quantification errors obtained when wrongly assuming homogeneous media were in the range +41 to +94% (P2), 0.1 to −7% (P3), and −39 to +44% (P4). Using a heterogeneous model, the overall error ranged from −7 to 7%. In conclusion, this work demonstrates that assuming homogeneous media leads to noticeable quantification errors that can be improved by adopting heterogeneous models.

Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung. Ex vivo MPM imaging of intrinsic two-photon excited fluorescence was performed on mouse and canine neoplastic, inflammatory and tumor-free lung sites. Results showed that MPM detected microanatomical differences between tumor-free and neoplastic lung tissue similar to standard histopathology but without the need for tissue processing. Furthermore, inflammatory sites displayed a distinct red-shifted fluorescence compared to neoplasms in both mouse and canine lung, and adenocarcinomas displayed a less pronounced fluorescence emission in the 500 to 550 nm region compared to adenomas in mouse models of lung cancer. These spectral distinctions were also confirmed by two-photon excited fluorescence microspectroscopy. We demonstrate the feasibility of applying MPM imaging of intrinsic fluorescence for the differentiation of lung neoplasms, inflammatory and tumor-free lung, which motivates the application of multiphoton endoscopy for the in situ imaging of lung nodules.

Accurate quantification of circulating cell populations in mice is important in many areas of preclinical biomedical research. Normally, this is done either by extraction and analysis of small blood samples or, more recently, by using microscopy-based in vivo fluorescence flow cytometry. We describe a new technological approach to this problem using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The diffuse fluorescence flow cytometer (DFFC) uses a laser to illuminate a mouse limb and an array of optical fibers coupled to a high-sensitivity photomultiplier tube array operating in photon counting mode to detect weak fluorescence signals from cells. We first demonstrate that the DFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD-labeled cells in a custom-made optical flow phantom with similar size, optical properties, linear flow rates, and autofluorescence as a mouse limb. We also present preliminary data demonstrating that the DFFC is capable of detecting circulating cells in nude mice in vivo. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with sensitivity improvement by several orders of magnitude compared to current approaches.

Design strategies for high-sensitivity refractive index sensors based on the principle of wavelength-selective resonant coupling in dual-core photonic crystal fibers are presented. Phase matching at a single wavelength can be achieved between an analyte-filled microstructured core and a small core with a down-doped rod or one small air hole in the center, thus enabling selectively directional resonant-coupling between the two cores. The transmission spectra of the output light presents a notch at the index-matched wavelength, yielding a resonant wavelength depending on the refractive index of the analyte. Numerical simulations demonstrate that both of the two proposed sensors can be used for highly sensitive detection of low-index analyte. In particular, the configuration realized by introducing the fiber with a small air hole in one core can be used to the detection of the analyte index around 1.33 and the sensitivity reach to 1.2×10⁴  nm per refractive index unit (RIU). In addition, the detection limit is as low as 2.5×10−7  RIU at na = 1.33.

Surface-enhanced Raman spectroscopy (SERS) has been shown to be able to detect low-concentration biofluids. Saliva SERS readings of 21 lung cancer patients and 20 normal people were measured and differentiated. Most of the Raman peak intensities decrease for lung cancer patients compared with that of normal people. Those peaks were assigned to proteins and nucleic acids, which indicate a corresponding decrease of those substances in saliva. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to reduce and discriminate between the two groups of data, and the study resulted in accuracy, sensitivity, and specificity being 80%, 78%, and 83%, respectively. In conclusion, SERS of saliva showed the ability to predict lung cancer in our experiment.

Evanescent field-fiber loop ringdown (EF-FLRD) is a relatively new hybrid sensing technique which combines a versatile sensing mechanism with a sensitivity-enhanced ringdown detection scheme. An array of low cost, fast response, and high sensitivity biosensors based on the EF-FLRD technique can be developed. In this work, new fiber loop ringdown glucose sensors using refractive index-difference evanescent field attenuation effect as a sensing mechanism are described. The sensor head consists of either a section of partially-etched bare single mode fiber or a section of the etched fiber with glucose oxidase (GOD) immobilized on the etched fiber surface. Effects of the sensor head, with and without the immobilized GOD, on the sensor's performance are comparatively examined. The sensors' responses to standard glucose solutions and synthetic urines in different glucose concentrations ranging from 50  mg/dl to 10  g/dl are studied. The sensors, with or without the immobilized GOD, showed a linear response to glucose concentrations in the range of 100  mg/dl to 1  g/dl, but a nonlinear response in the higher glucose concentration ranging from 1 to 10  g/dl. The detection sensitivities of the sensors for the glucose solutions and artificial urine samples are 75 and 50  mg/dl respectively, and the sampling rate of the sensors is 10 to 100 Hz. Estimated theoretical detection sensitivity of the EF-FLRD glucose sensors is 10  mg/dl, which is approximately 17 times lower than the glucose renal threshold concentration.

Imaging photoplethysmography (PPG) is able to capture useful physiological data remotely from a wide range of anatomical locations. Recent imaging PPG studies have concentrated on two broad research directions involving either high-performance cameras and or webcam-based systems. However, little has been reported about the difference between these two techniques, particularly in terms of their performance under illumination with ambient light. We explore these two imaging PPG approaches through the simultaneous measurement of the cardiac pulse acquired from the face of 10 male subjects and the spectral characteristics of ambient light. Measurements are made before and after a period of cycling exercise. The physiological pulse waves extracted from both imaging PPG systems using the smoothed pseudo-Wigner-Ville distribution yield functional characteristics comparable to those acquired using gold standard contact PPG sensors. The influence of ambient light intensity on the physiological information is considered, where results reveal an independent relationship between the ambient light intensity and the normalized plethysmographic signals. This provides further support for imaging PPG as a means for practical noncontact physiological assessment with clear applications in several domains, including telemedicine and homecare.

In this paper a simple photonic crystal fiber (PCF) interferometric breathing sensor is introduced. The interferometer consists of a section of PCF fusion spliced at the distal end of a standard telecommunications optical fiber. Two collapsed regions in the PCF caused by the splicing process allow the excitation and recombination of a core and a cladding PCF mode. As a result, the reflection spectrum of the device exhibits a sinusoidal interference pattern that instantly shifts when water molecules, present in exhaled air, are adsorbed on or desorbed from the PCF surface. The device can be used to monitor a person's breathing whatever the respiration rate. The device here proposed could be particularly important in applications where electronic sensors fail or are not recommended. It may also be useful in the evaluation of a person's health and even in the diagnosis and study of the progression of serious illnesses such as sleep apnea syndrome.

Diffuse correlation spectroscopy (DCS) is a novel optical technique that appears to be an excellent tool for assessing cerebral blood flow in a continuous and non-invasive manner at the bedside. We present new clinical validation of the DCS methodology by demonstrating strong agreement between DCS indices of relative cerebral blood flow and indices based on phase-encoded velocity mapping magnetic resonance imaging (VENC MRI) of relative blood flow in the jugular veins and superior vena cava. Data were acquired from 46 children with single ventricle cardiac lesions during a hypercapnia intervention. Significant increases in cerebral blood flow, measured both by DCS and by VENC MRI, as well as significant increases in oxyhemoglobin concentration, and total hemoglobin concentration, were observed during hypercapnia. Comparison of blood flow changes measured by VENC MRI in the jugular veins and by DCS revealed a strong linear relationship, R = 0.88, p<0.001, slope = 0.91±0.07. Similar correlations were observed between DCS and VENC MRI in the superior vena cava, R = 0.77, slope = 0.99±0.12, p<0.001. The relationship between VENC MRI in the aorta and DCS, a negative control, was weakly correlated, R = 0.46, slope = 1.77±0.45, p<0.001.

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.

We present a method for the determination of the absorption coefficient of turbid media in a broad wavelength range with high spectral resolution using a dual step method. First, the reduced scattering coefficient is determined for a few wavelengths with spatially resolved reflectance measurements. The reduced scattering coefficient for the intermediate wavelengths is interpolated by fitting a power law. Second, the absorption coefficient is obtained from measurements of the total reflectance using the a priori knowledge of the reduced scattering coefficient. By applying a white light source and a spectrometer to measure the total reflectance, the absorption coefficient is determined with a high spectral resolution. The methodology is verified by comparing the absorption coefficients determined by the spatially resolved reflectance measurements with those obtained by the dual step method. The influence of an unknown refractive index and phase function on the determination of the optical properties is investigated. In addition, the optical properties of Intralipid/ink phantoms and the fat layer of porcine rind were determined. The absorption coefficient of the investigated phantoms varying by four orders of magnitude could be determined with an average error of less than 10%.

A homogeneous illumination of intra-abdominal organs is essential for successful photodynamic therapy of the abdominal cavity. Considering the current lack of outstanding light-delivery systems, a new illumination procedure was assessed. A rat model of peritoneal carcinomatosis was used. Four hours after intraperitoneal injection of hexaminolevulinate, a square illuminating panel connected to a 635-nm laser source was inserted vertically into the abdominal cavity. The abdominal incision was sutured and a pneumoperitoneum created prior to illumination. Light dosimetry was based on the calculation of the peritoneal surface by MRI. The rats were treated with a light dose of 20, 10, 5 or 2.5  J/cm² administered continuously with an irradiance of 7  mW/cm². The homogeneity of the cavity illumination was assessed by quantification of the photobleaching of the tumor lesions according to their localization and by scoring of that of the liver and of the bowel immediately after treatment. Photobleaching quantification for tumor lesions relied on the calculation of the fluorescence intensity ratio (after/before treatment) after recording of the lesions during blue-light laparoscopy and determination of their fluorescence intensity with Sigmascan Pro software. The procedure led to a homogeneous treatment of the abdominal cavity. No statistical difference was observed for the photobleaching values according to the localization of the lesions on the peritoneum (p = 0.59) and photobleaching of the liver and of the intestine was homogeneous. We conclude that this procedure can successfully treat the major sites involved in peritoneal carcinomatosis.

Vocal fold scarring is a predominant cause of voice disorders yet lacks a reliable treatment method. The injection of soft biomaterials to improve mechanical compliance of the vocal folds has emerged as a promising treatment. Here, we study the use of precise femtosecond laser microsurgery to ablate subsurface voids, with a goal of eventually creating a plane in dense subepithelial scar tissue into which biomaterials can be injected for their improved localization. Specifically, we demonstrate the ablation of small subepithelial voids in porcine vocal fold tissue up to 120 µm below the surface such that larger voids in the active area of vocal fold mucosa ( ∼ 3×10  mm²) can eventually be ablated in about 3 min. We use sub-µJ, 776-nm pulses from a compact femtosecond fiber laser system operating at a 500-kHz repetition rate. The use of relatively high repetition rates, with a small number of overlapping pulses, is critical to achieving ablation in a very short time while still avoiding significant heat deposition. Additionally, we use the same laser for nonlinear optical imaging to provide visual feedback of tissue structure and to confirm successful ablation. The ablation parameters, including pulse duration, pulse energy, spot size, and scanning speed, are comparable to the specifications in our recently developed miniaturized femtosecond laser surgery probes, illustrating the feasibility of developing an ultrafast laser surgical instrument.

The theory of hard tissue ablation reported for IR lasers is based on a process of thermomechanical interaction, which is explained by the absorption of the radiation in the water component of the tissue. The microexplosion of the water is the cause of tissue fragments being blasted from hard tissue. The aim of this study is to evaluate the influence of the interdependence of water layer thickness and incident radiant exposure on ablation performance. A total of 282 specimens of bovine shank bone were irradiated with a pulse CO₂ laser. Irradiation was carried out in groups: without a water layer and with a static water layer of thickness ranging from 0.2 to 1.2 mm. Each group was subdivided into five subgroups for different radiant exposures ranging from 18 to 84  J/cm², respectively. The incision geometry, surface morphology, and microstructure of the cut walls as well as thermal injury were examined as a function of the water layer thickness at different radiant exposures. Our results demonstrate that the additional water layer is actually a mediator of laser-tissue interaction. There exists a critical thickness of water layer for a given radiant exposure, at which the additional water layer plays multiple roles, not only acting as a cleaner to produce a clean cut but also as a coolant to prevent bone heating and reduce thermal injury, but also helping to improve the regularity of the cut shape, smooth the cut surface, and enhance ablation rate and efficiency. The results suggest that desired ablation results depend on optimal selection of both water layer thickness and radiant exposure.

A book review of a recently released title.