We show that myelin, the insulation wrap of nerve fibers, can couple laser light, thus behaving as a single-cell optical device. The effect was employed to map distinct myelin regions based on the coupling efficiency. Raman spectra acquisition allowed us to simultaneously understand the underlying microscopic differences in the membrane lipid ordering degree. The described method potentially provides new capabilities in myelin-associated disease studies and can be used as a handy tool for myelin structure investigation in combination with other methods.

Cyclooxygenase-2 (COX-2) is a promising target for the imaging of cancer in a range of diagnostic and therapeutic settings. We report a near-infrared COX-2-targeted probe, fluorocoxib C (FC), for visualization of solid tumors by optical imaging. FC exhibits selective and potent COX-2 inhibition in both purified protein and human cancer cell lines. <italic<In vivo</italic< optical imaging shows selective accumulation of FC in COX-2-overexpressing human tumor xenografts [1483 head and neck squamous cell carcinoma (HNSCC)] implanted in nude mice, while minimal uptake is detectable in COX-2-negative tumor xenografts (HCT116) or 1483 HNSCC xenografts preblocked with the COX-2-selective inhibitor celecoxib. Time course imaging studies conducted from 3 h to 7-day post-FC injection revealed a marked reduction in nonspecific fluorescent signals with retention of fluorescence in 1483 HNSCC tumors. Thus, use of FC in a delayed imaging protocol offers an approach to improve imaging signal-to-noise that should improve cancer detection in multiple preclinical and clinical settings.

The concept of snapshot red-green-blue (RGB) multispectral imaging was applied for skin chromophore mapping. Three monochromatic spectral images have been extracted from a single RGB image dataset at simultaneous illumination of skin by 473-, 532-, and 659-nm laser lines. The spectral images were further transformed into distribution maps of skin melanin, oxyhemoglobin, and deoxyhemoglobin, related to pigmented and vascular skin malformations. The performance and clinical potential of the proposed technique are discussed.

Lymph nodes play a central role in metastatic cancer spread and are a key clinical assessment target. Abnormal node vascularization, morphology, and size may be indicative of disease but can be difficult to visualize with sufficient accuracy using existing clinical imaging modalities. To explore the potential utility of photoacoustic imaging for the assessment of lymph nodes, images of <italic<ex vivo</italic< samples were obtained at multiple wavelengths using a high-resolution three-dimensional photoacoustic scanner. These images showed that hemoglobin based contrast reveals nodal vasculature and lipid-based contrast reveals the exterior node size, shape, and boundary integrity. These two sources of complementary contrast may allow indirect observation of cancer, suggesting a future role for photoacoustic imaging as a tool for the clinical assessment of lymph nodes.

Test-retest reliability of neuroimaging measurements is an important concern in the investigation of cognitive functions in the human brain. To date, intraclass correlation coefficients (ICCs), originally used in inter-rater reliability studies in behavioral sciences, have become commonly used metrics in reliability studies on neuroimaging and functional near-infrared spectroscopy (fNIRS). However, as there are six popular forms of ICC, the adequateness of the comprehensive understanding of ICCs will affect how one may appropriately select, use, and interpret ICCs toward a reliability study. We first offer a brief review and tutorial on the statistical rationale of ICCs, including their underlying analysis of variance models and technical definitions, in the context of assessment on intertest reliability. Second, we provide general guidelines on the selection and interpretation of ICCs. Third, we illustrate the proposed approach by using an actual research study to assess intertest reliability of fNIRS-based, volumetric diffuse optical tomography of brain activities stimulated by a risk decision-making protocol. Last, special issues that may arise in reliability assessment using ICCs are discussed and solutions are suggested.

This PDF file contains the editorial “Special Section Guest Editorial: Laser Applications in Life Sciences” for JBO Vol. 20 Issue 05

The study reports an investigation of the photoproducts obtained by exposure of chlorpromazine hydrochloride in ultrapure water (concentration 2  mg/mL) to a 266-nm laser beam obtained by fourth harmonic generation from a Nd:YAG laser (6-ns full time width at half maximum, 10-Hz pulse repetition rate). The photoproducts were analyzed by steady-state UV-Vis absorption, laser-induced fluorescence, Fourier transform infrared spectroscopy, and liquid chromatography–tandem time-of-flight mass spectroscopy. Two figures showing pathways that take place during irradiation for obtaining the final products are shown. The quantum yield of singlet oxygen generation by chlorpromazine (CPZ) was determined relative to standard Zn-phthalocyanine in dimethyl sulfoxide. To outline the role of fluorescence in photoproducts formation rates, fluorescence quantum yield of CPZ during exposure to 355-nm radiation (third harmonic of the fundamental beam of Nd:YAG laser) was investigated relative to standard Coumarin 1 in ethanol. The CPZ solutions exposed 60 and 240 min to 266-nm laser beam, respectively, were tested against Staphylococcus aureus ATCC 25923 strain. For 25  μL of CPZ samples irradiated 240 min, a higher diameter of inhibition has obtained against the tested strain than for the 60-min exposed ones.

While much is known about the effect of smoke and vapors on the composition of blood, little is known about their impact on the composition of breath. When tobacco from traditional cigarettes (T) is burned, it produces harmful smoke compared with the vapor produced when using electronic cigarettes (E). Using a noninvasive, safe, and rapid CO² laser-photoacoustic method, this study aimed to examine the ethylene changes at different time intervals in the exhaled breath composition of E-cigarette smokers and T-cigarette smokers, before and after the consecutive exposures to cigarettes. Oxidative stress from exposure to tobacco smoke has a role in the pathogenic process, leading to chronic obstructive pulmonary disease. The evidence on the mechanisms by which T-smoking causes damage indicates that there is no risk-free level of exposure to tobacco smoke. The study revealed that the ethylene level (in the E-cigarette smoker’s case) was found to be in smaller concentrations (compared with T-cigarette smoker’s case) and that E-cigarettes may provide an alternative to T-cigarette smoking.

The photoisomerization of relatively nontoxic E-combretastatins to clinically active Z-isomers is shown to occur in solution through both one- and two-photon excitations at 340 and 625 nm, respectively. The photoisomerization is also demonstrated to induce mammalian cell death by a two-photon absorption process at 625 nm. Unlike conventional photodynamic therapy (PDT), the mechanism of photoisomerization is oxygen-independent and active in hypoxic environments such as in tumors. The use of red or near-infrared (NIR) light for two-photon excitation allows greater tissue penetration than conventional UV one-photon excitation. The results provide a baseline for the development of a novel phototherapy that overcomes nondiscriminative systemic toxicity of Z-combretastatins and the limitations of PDT drugs that require the presence of oxygen to promote their activity, with the added benefits of two-photon red or NIR excitation for deeper tissue penetration.

Gold nanoparticles are delivered into living cells by transient electroporation method to obtain intracellular surface-enhanced Raman spectroscopy (SERS). The subcellular localization of gold nanoparticles is characterized by transmission electron microscopy, and the forming large gold nanoaggregates are mostly found in the cytoplasm. The SERS detection of cells indicates that this kind of gold nanostructures induces a high signal enhancement of cellular chemical compositions, in addition to less cellular toxicity than that of silver nanoparticles. These results demonstrate that rapid incorporation of gold nanoparticles by electroporation into cells has great potential applications in the studies of cell biology and biomedicine.

In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs’ signal intensity from the skin surface to a depth of 4  μm. Below 4  μm, the Ag NPs’ signal continues to decline, having completely disappeared at 12 to 14  μm depth. CRM shows that the penetration depth of Ag NPs is 11.1±2.1  μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6±8.3  μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

Photodynamic therapy (PDT) has become a promising alternative for treatment of skin lesions such as squamous cell carcinoma. We propose a method to monitor the effects of PDT in a noninvasive way by using the optical attenuation coefficient (OAC) calculated from optical coherence tomography (OCT) images. We conducted a study on mice with chemically induced neoplastic lesions and performed PDT on these lesions using homemade photosensitizers. The response of neoplastic lesions to therapy was monitored using, at the same time, macroscopic clinical visualization, histopathological analysis, OCT imaging, and OCT-based attenuation coefficient measurement. Results with all four modalities demonstrated a positive response to treatment. The attenuation coefficient was found to be 1.4 higher in skin lesions than in healthy tissue and it decreased after therapy. This study shows that the OAC is a potential tool to noninvasively assess the evolution of skin neoplastic lesions with time after treatment.

There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology. We have evaluated the potential of a visible ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvants. Specifically, we demonstrate that vaccination of mice with laser-inactivated H1N1 influenza virus at about a 10-fold lower dose than that required using conventional formalin-inactivated influenza vaccines results in protection against lethal H1N1 challenge in mice. The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay. Unlike conventional inactivation methods, laser treatment did not generate carbonyl groups in protein, thereby reducing the risk of adverse vaccine-elicited T helper type 2 responses. Therefore, USP laser treatment is an attractive potential strategy to generate WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques.

Colloid response is a type of tumor response that occurs after preoperative radiochemotherapy for rectal carcinoma. Given its important influence on survival, the colloid response should be considered when estimating histopathological reactions. Here, multiphoton microscopy (MPM) was applied to evaluate the colloid response <italic<ex vivo</italic<. This study demonstrated that MPM has the capability to visualize the colloid response in the absence of labels and can, in particular, identify rare residual carcinomatous cells in mucin pools. These results highlight the potential of this nonlinear optical technique as a diagnostic tool for tumor response after neoadjuvant treatment.

In the cardiovascular system, the macrocirculation and microcirculation—two subsystems—can be affected by aging. Laser speckle contrast imaging (LSCI) is an emerging noninvasive optical technique that allows the monitoring of microvascular function and can help, using specific data processing, to understand the relationship between the subsystems. Using LSCI, the goals of this study are: (i) to assess the aging effect over microvascular parameters (perfusion and moving blood cells velocity, MBCV) and macrocirculation parameters (pulse-wave velocity, PWV) and (ii) to study the relationship between these parameters. In 16 healthy subjects (20 to 62 years old), perfusion and MBCV computed from LSCI are studied in three physiological states: rest, vascular occlusion, and post-occlusive reactive hyperaemia (PORH). MBCV is computed from a model of velocity distribution. During PORH, the experimental results show a relationship between perfusion and age (R2=0.67) and between MBCV and age (R2=0.72), as well as between PWV and age at rest (R2=0.91). A relationship is also found between perfusion and MBCV for all physiological states (R2=0.98). Relationships between microcirculation and macrocirculation (perfusion-PWV or MBCV-PWV) are found only during PORH with R2=0.76 and R2=0.77, respectively. This approach may prove useful for investigating dysregulation in blood flow.

Hair follicle offers an excellent model for systems biology and regenerative medicine. So far, the stages of hair follicle growth have been evaluated by histological examination. In this work, a noninvasive spectroscopy was proposed by measuring the diffuse reflectance of mouse skin and analyzing the melanin value. Results show that the skin diffuse reflectance was relatively high when hair follicles were at the telogen stage and at the beginning of the anagen stage, and decreased with the progression of the anagen stage. When the hair follicle entered into the catagen stage, the diffuse reflectance gradually increased. The changes in the melanin content of skin had contrary dynamics. Substages of the hair follicle cycle could be distinguished by comparing the changes in melanin value with the histological examination. This study provided a new method for noninvasive evaluation of the hair follicle stage, and should be valuable for basic and therapeutic investigations on hair regeneration.

We demonstrate a multimodal approach that combines a pump–probe with confocal reflectance and multiphoton autofluorescence microscopy. Pump–probe microscopy has been proven to be of great value in analyzing thin tissue sections of pigmented lesions, as it produces molecular contrast which is inaccessible by other means. However, the higher optical intensity required to overcome scattering in thick tissue leads to higher-order nonlinearities in the optical response of melanin (e.g., two-photon pump and one-photon probe) that present additional challenges for interpreting the data. We show that analysis of pigment composition in vivo must carefully account for signal terms that are nonlinear with respect to the pump and probe intensities. We find that pump–probe imaging gives useful contrast for pigmented structures over a large range of spatial scales (100  μm to 1 cm), making it a potentially useful tool for tracking the progression of pigmented lesions without the need to introduce exogenous contrast agents.

Atopic dermatitis (AD) is a multifactorial inflammatory skin disease that affects both children and adults in an increasing manner. The treatment of AD often reduces subjective skin parameters, such as itching, dryness, and tension, but the inflammation cannot be cured. Laser scanning microscopy was used to investigate the skin surface, epidermal, and dermal characteristics of dry and atopic skin before and after treatment with an ointment rich in hyperforin, which is known for its anti-inflammatory effects. The results were compared to subjective parameters and transepidermal water loss, stratum corneum moisture, and stratum corneum lipids. Using biophysical methods, in particular laser scanning microscopy, it was found that atopic skin has distinct features compared to healthy skin. Treatment with a hyperforin-rich ointment resulted in an improvement of the stratum corneum moisture, skin surface dryness, skin lipids, and the subjective skin parameters, indicating that the barrier is stabilized and improved by the ointment. But in contrast to the improved skin surface, the inflammation in the deeper epidermis/dermis often continues to exist. This could be clearly shown by the reflectance confocal microscopy (RCM) measurements. Therefore, RCM measurements could be used to investigate the progress in treatment of atopic dermatitis.

We investigate the antibacterial effect of ultrafine nanodiamond particles with an average size of 5 nm against the gram-negative bacteria Escherichia coli (E. coli). UV-visible, Raman spectroscopy, and scanning electron microscopy (SEM) have been employed to elucidate the nature of the interaction. The influence on bacterial growth was monitored by measuring optical densities of E. coli at 600 nm as a function of time in the presence of carboxylated nanodiamond (cND) particles (100  μg/ml) in highly nutritious liquid Luria–Bertani medium. The SEM images prove that cND particles are attached to the bacterial cell wall surface and some portion of the bacterial cell wall undergoes destruction. Due to the change of the protein structure on the bacterial wall, a small Raman shift in the region of 1400 to 1700  cm−¹ was observed when E. coli interacted with cNDs. Raman mapping images show strong evidence of cND attachment at the bacterial cell wall surface. Electrotransformation of E. coli with a fluorescent protein markers experiment demonstrated that the interaction mechanisms are different for E. coli treated with cND particles, E. coli by lysozyme treatment, and E. coli that suffer lysis.

The analysis of the structure-function relationship is extremely important in the study of proteins. The importance of function-related motions of large parts or subglobules of protein molecules stimulates the spectroscopic study in the low-frequency (terahertz) domain. However, only tentative assignments are available and the spectroscopic data are insufficiently discussed in terms of structural changes. This work is aimed at the analysis of regularities of changes in the low-frequency (100 to 600  cm⁻¹) FTIR and Raman spectra of proteins related to their structural modifications. We study the spectra of two proteins with substantially different structures (albumin and chymotrypsin) and the spectra of samples in which the structures of protein molecules are modified using inhibition, thermal denaturation, and cleavage of disulfide bonds. The results indicate that the low-frequency spectral interval can be used to characterize protein conformations. Correlated variations in the intensities of several low-frequency bands are revealed in the spectra of the modified proteins. The strongest spectral changes are caused by thermal denaturation of proteins, and the effect of cleavage of disulfide bonds is generally weaker. It is demonstrated that the inhibitor binding in the active site causes spectral changes that can be compared to the changes induced by thermal denaturation.

Multiphoton microscopy has become essential for dynamic imaging in thick living tissues. High-rate, full-field image acquisition in multiphoton microscopy is achievable by parallelization of the excitation and detection pathways. We developed our approach via a diffractive optical element which splits a pulsed laser into 16 beamlets and exploits a descanned detection system consisting of an array of beamlet-associated photomultiplier tubes. The optical performance of the multiphoton multispot system (MCube) has been characterized in cardiac tissue sections and subsequently used for the first time for fluorescence imaging of cardiomyocyte Ca²+ dynamics in viable acute cardiac slices. Multispot multiphoton microscopy (MMM) has never been used before to monitor Ca²+ dynamics in thick, viable tissue samples. Acute heart slices are a powerful close-to-in vivo model of Ca²+ imaging allowing the simultaneous observation of several cells in their own tissue environment, exploiting the multiphoton excitation ability to penetrate scattering tissues. Moreover, we show that the concurrent high spatial and temporal resolutions afforded by the parallel scanning in MMM can be exploited to simultaneously assess subcellular Ca²+ dynamics in different cells in the tissue. We recorded local Ca²+ release events including macrosparks, travelling waves, and rotors.

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

Ultrasmall photoluminescent gold nanoclusters (Au NCs), composed of several atoms with sizes up to a few nanometers, have recently stimulated extensive interest. Unique molecule-like behaviors, low toxicity, and facile synthesis make photoluminescent Au NCs a very promising alternative to organic fluorophores and semiconductor quantum dots (QDs) in broad ranges of biomedical applications. However, using gold nanoparticles (Au NPs) for bioimaging might cause their degradation under continuous excitation with UV light, which might result in toxicity. We report spectral changes of photoluminescent 2-(N-morpholino) ethanesulfonic acid (MES)-coated (Au-MES) NCs under irradiation with UV/blue light. Photoluminescent water soluble Au-MES NCs with a photoluminescence (PL) band maximum at 476 nm (λex=420  nm) were synthesized. Under irradiation with 402 nm wavelength light the size of photoluminescent Au-MES NCs decreased (λem=430  nm). Irradiating the sample solution with 330 nm wavelength light, nonluminescent Au NPs were disrupted, and photoluminescent Au NCs (λem=476  nm) were formed. Irradiation with 330 nm wavelength light did not directly affect photoluminescent Au-MES NCs, however, increase in PL intensity indicated the formation of photoluminescent Au NCs from the disrupted nonluminescent Au NPs. This study gives a good insight into the photostability of MES-coated Au NPs under continuous excitation with UV/blue light.

Part of the optical clearing study in biological tissues concerns the determination of the diffusion characteristics of water and optical clearing agents in the subject tissue. Such information is sufficient to characterize the time dependence of the optical clearing mechanisms—tissue dehydration and refractive index (RI) matching. We have used a simple method based on collimated optical transmittance measurements made from muscle samples under treatment with aqueous solutions containing different concentrations of ethylene glycol (EG), to determine the diffusion time values of water and EG in skeletal muscle. By representing the estimated mean diffusion time values from each treatment as a function of agent concentration in solution, we could identify the real diffusion times for water and agent. These values allowed for the calculation of the correspondent diffusion coefficients for those fluids. With these results, we have demonstrated that the dehydration mechanism is the one that dominates optical clearing in the first minute of treatment, while the RI matching takes over the optical clearing operations after that and remains for a longer time of treatment up to about 10 min, as we could see for EG and thin tissue samples of 0.5 mm.

The ability to conduct highly localized delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, and signaling molecules or dyes to live mammalian embryos is greatly desired to enable a variety of studies in the field of developmental biology, such as investigating the molecular regulation of cardiovascular morphogenesis. To meet such a demand, we introduce, for the first time, the concept of employing optical coherence tomography (OCT)-guide microinjections in live mouse embryos, which provides precisely targeted manipulation with spatial resolution at the micrometer scale. The feasibility demonstration is performed with experimental studies on cultured live mouse embryos at E8.5 and E9.5. Additionally, we investigate the OCT-guided microinjection of gold–silica nanoshells to the yolk sac vasculature of live cultured mouse embryos at the stage when the heart just starts to beat, as a potential approach for dynamic assessment of cardiovascular form and function before the onset of blood cell circulation. Also, the capability of OCT to quantitatively monitor and measure injection volume is presented. Our results indicate that OCT-guided microinjection could be a useful tool for mouse embryonic research.

Photoacoustic imaging (PAI) with a linear-array-based probe can provide a convenient means of imaging the human microcirculation within its native structural context and adds functional information. PAI using a multielement linear transducer array combined with multichannel collecting system was used for in vivo volumetric imaging of the blood microcirculation, the total concentration of hemoglobin (HbT), and the hemoglobin oxygen saturation (sO₂) within human tissue. Three-dimensional (3-D) PA and ultrasound (US) volumetric scans were acquired from the forearm skin by linearly translating the transducer with a stepper motor over a region of interest, while capturing two-dimensional images using 15, 21, and 40 MHz frequency transducer probes. For the microvasculature imaging, PA images were acquired at 800- and 1064-nm wavelengths. For the HbT and sO₂ estimates, PA images were collected at 750- and 850-nm wavelengths. 3-D microcirculation, HbT, and sO₂ maps of the forearm skin were obtained from normal subjects. The linear-array-based PAI has been found promising in terms of resolution, imaging depth, and imaging speed for in vivo microcirculation imaging within human skin. We believe that a reflection type probe, similar to existing clinical US probes, is most likely to succeed in real clinical applications. Its advantages include ease of use, speed, and familiarity for radiographers and clinicians.

Low level light therapy (LLLT) has numerous therapeutic benefits, including improving wound healing, but the precise mechanisms involved are not well established; in particular, the underlying role of cytochrome C oxidase (C-ox) as the primary photoacceptor and the associated biochemical mechanisms still require further investigation. We previously showed the nitric oxide (NO) donating drug nitrosyl-cobinamide (NO-Cbi) enhances wound healing through a cGMP/cGMP-dependent protein kinase/ERK1/2 mechanism. Here, we show that the combination of LLLT and NO-Cbi markedly improves wound healing compared to either treatment alone. LLLT-enhanced wound healing proceeded through an electron transport chain-C-ox-dependent mechanism with a reduction of reactive oxygen species and increased adenosine triphosphate production. C-ox was validated as the primary photoacceptor by three observations: increased oxygen consumption, reduced wound healing in the presence of sodium azide, and disassociation of cyanide, a known C-ox ligand, following LLLT. We conclude that LLLT and NO-Cbi accelerate wound healing through two independent mechanisms, the electron transport chain-C-ox pathway and cGMP signaling, respectively, with both resulting in ERK1/2 activation.

Less invasive methods for treating dental caries are strongly desired. However, conventional dental lasers do not always selectively remove caries or ensure good bonding to the composite resin. According to our previous study, demineralized dentin might be removed by a nanosecond pulsed laser operating at wavelengths of around 5.8  μm. The present study investigated the irradiation effect of the light on carious human dentin classified into “remove,” “not remove,” and “unclear” categories. Under 5.85-μm laser pulses, at average power densities of 30  W/cm² and irradiation time of 2 s, the ablation depth of “remove” and “not remove,” and also the ablation depth of “unclear” and “not remove,” were significantly different (p<0.01). The ablation depth was correlated with both Vickers hardness and Ca content. Thus, a nanosecond pulsed laser operating at 5.85  μm proved an effective less-invasive caries treatment.

Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing <italic<in vitro</italic< collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a “clean” environment, free of the myriad cells, and signals found in <italic<in vivo</italic<. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility.

We present a compact fluorescence imaging system developed for real-time sentinel lymph node mapping. The device uses two near-infrared wavelengths to record fluorescence and anatomical images with a single charge-coupled device camera. Experiments on lymph node and tissue phantoms confirmed that the amount of dye in superficial lymph nodes can be better estimated due to the absorption correction procedure integrated in our device. Because of the camera head’s small size and low weight, all accessible regions of tissue can be reached without the need for any adjustments.

n order to estimate multispectral images of the absorption and scattering properties in the cerebral cortex of in vivo rat brain, we investigated spectral reflectance images estimated by the Wiener estimation method using a digital RGB camera. A Monte Carlo simulation-based multiple regression analysis for the corresponding spectral absorbance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) was then used to specify the absorption and scattering parameters of brain tissue. In this analysis, the concentrations of oxygenated hemoglobin and that of deoxygenated hemoglobin were estimated as the absorption parameters, whereas the coefficient a and the exponent b of the reduced scattering coefficient spectrum approximated by a power law function were estimated as the scattering parameters. The spectra of absorption and reduced scattering coefficients were reconstructed from the absorption and scattering parameters, and the spectral images of absorption and reduced scattering coefficients were then estimated. In order to confirm the feasibility of this method, we performed in vivo experiments on exposed rat brain. The estimated images of the absorption coefficients were dominated by the spectral characteristics of hemoglobin. The estimated spectral images of the reduced scattering coefficients had a broad scattering spectrum, exhibiting a larger magnitude at shorter wavelengths, corresponding to the typical spectrum of brain tissue published in the literature. The changes in the estimated absorption and scattering parameters during normoxia, hyperoxia, and anoxia indicate the potential applicability of the method by which to evaluate the pathophysiological conditions of in vivo brain due to the loss of tissue viability.

This work reports that the laser fluence rate inside porcine skin varied notably with the change of tissue water content under the same laser irradiation conditions. The laser fluence rate inside skin tissue samples with varying water content was measured using an optical fiber sensor, while the target was irradiated either by a low-level 635 or 830 nm laser (50  mW/cm²). It was demonstrated that the distribution of laser fluence rate inside the target is strongly affected by tissue water content and its profile is determined by the water content dependency of optical properties at the laser wavelength.

The eardrum or tympanic membrane (TM) transforms acoustic energy at the ear canal into mechanical motions of the ossicles. The acousto-mechanical transformer behavior of the TM is determined by its shape, three-dimensional (3-D) motion, and mechanical properties. We have developed an optoelectronic holographic system to measure the shape and 3-D sound-induced displacements of the TM. The shape of the TM is measured with dual-wavelength holographic contouring using a tunable near IR laser source with a central wavelength of 780 nm. 3-D components of sound-induced displacements of the TM are measured with the method of multiple sensitivity vectors using stroboscopic holographic interferometry. To accurately obtain sensitivity vectors, a new technique is developed and used in which the sensitivity vectors are obtained from the images of a specular sphere that is being illuminated from different directions. Shape and 3-D acoustically induced displacement components of cadaveric human TMs at several excitation frequencies are measured at more than one million points on its surface. A numerical rotation matrix is used to rotate the original Euclidean coordinate of the measuring system in order to obtain in-plane and out-of-plane motion components. Results show that in-plane components of motion are much smaller (<20%) than the out-of-plane motions’ components.

Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A₃ adenosine receptors (A₃R) were blocked by A₃R antagonist compared with A₁ adenosine receptor or A_2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors’ expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI.

This paper discusses one of the key problems of laser-induced tissue/cell hyperthermia mediated by gold nanoparticles, namely, quantifying and precise prediction of the light exposure to provide a controllable local heating impact on living organisms. The distributions of such parameters as an efficiency factor of absorption, differential and integral absorbing power of a nanoparticle, temperature increment, and Arrhenius damage integral were used to quantify nanoparticle effectiveness in the two-dimensional coordinate space “laser wavelength (λ)× radius of gold nanoparticles (R).” It was found that the fulfillment of required spatial and temporal characteristics of temperature fields in the vicinity of nanoparticle determines the optimal λ and R. As a result, the area in the space (λ×R) with a minimal criticality to alterations of the local hyperthermia may be significantly displaced from the position of the plasmonic resonance. The aspects of generalization of the proposed methodology for the analysis of local hyperthermia using nanoparticles of different shapes (nanoshells, nanorods, nanostars) and short pulse laser radiation are discussed.

The autofluorescence lifetime of healthy human skin was measured using excitation provided by a picosecond diode laser operating at a wavelength of 405 nm and with fluorescence emission collected at 475 and 560 nm. In addition, spectral and temporal responses of healthy human skin and intradermal nevus in the spectral range 460 to 610 nm were studied before and after photobleaching. A decrease in the autofluorescences lifetimes changes was observed after photobleaching of human skin. A three-exponential model was used to fit the signals, and under this model, the most significant photoinduced changes were observed for the slowest lifetime component in healthy skin at the spectral range 520 to 610 nm and intradermal nevus at the spectral range 460 to 610 nm.

Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

Depth resolved coherence gating along with Doppler shift detection of the carrier frequency is used for one predetermined velocity mapping in different flows. Bidirectional rapid scanning optical delay of optical coherence tomography system is applied in the reference arm. Tilted capillary entry is used as a hydrodynamic phantom to model a sign-variable flow with complex geometry. Structural and one specific velocity images are obtained from the scanning interferometer signal processing in the frequency domain using analog and digital filtering. A standard structural image is decomposed into three parts: stationary object, and positive and negative velocity distributions. The latter two show equivelocity maps of the flow. The final image is represented as the complexation of the three.

This study combines several fluorescence detection methods to distinguish structural features of the synovium and cartilage tissues and to visualize the localization of endogenous porphyrins in the sensitized tissues. Specimens of synovium and cartilage tissues obtained from rabbits with antigen-induced monoarthritis after intra-articular 5-aminolevulinic acid methyl ester injection and those from healthy rabbits were investigated <italic<ex vivo</italic< by means of fluorescence spectroscopy, fluorescence intensity, and lifetime microscopy. The presence of endogenous porphyrins was confirmed with the fluorescence spectra measured on sliced sensitized specimens. Application of the lifetime-gating method on fast fluorescence lifetime imaging microscopy images, allowed separate visualization of tissue structures possessing different average lifetimes. The presence of the structures has been validated by histopathological imaging based on conventional rapid hematoxylin–eosin staining of the specimens. The fluorescence lifetime of endogenous protoporphyrin IX has been assessed and employed for visualization of sensitized tissues.

We present the development of a transcutaneous Raman spectroscopy system and analysis algorithm for noninvasive glucose sensing. The instrument and algorithm were tested in a preclinical study in which a dog model was used. To achieve a robust glucose test system, the blood levels were clamped for periods of up to 45 min. Glucose clamping and rise/fall patterns have been achieved by injecting glucose and insulin into the ear veins of the dog. Venous blood samples were drawn every 5 min and a plasma glucose concentration was obtained and used to maintain the clamps, to build the calibration model, and to evaluate the performance of the system. We evaluated the utility of the simultaneously acquired Raman spectra to be used to determine the plasma glucose values during the 8-h experiment. We obtained prediction errors in the range of ∼1.5−2  mM. These were in-line with a best-case theoretical estimate considering the limitations of the signal-to-noise ratio estimates. As expected, the transition regions of the clamp study produced larger predictive errors than the stable regions. This is related to the divergence of the interstitial fluid (ISF) and plasma glucose values during those periods. Two key contributors to error beside the ISF/plasma difference were photobleaching and detector drift. The study demonstrated the potential of Raman spectroscopy in noninvasive applications and provides areas where the technology can be improved in future studies.

Femtosecond near-infrared lasers are widely used for a number of ophthalmic procedures, with flap cutting in the laser-assisted in situ keratomileusis (LASIK) surgery being the most frequent one. At the same time, lasers of this type, equipped with harmonic generators, have been shown to deliver enough ultraviolet (UV) power for the second stage of the LASIK procedure, the stromal ablation. However, the speed of the ablation reported so far was well below the currently accepted standards. Our purpose was to perform high-speed photorefractive keratectomy (PRK) with femtosecond UV pulses in rabbits and to evaluate its predictability, reproducibility and healing response. The laser source delivered femtosecond 206 nm pulses with a repetition rate of 50 kHz and an average power of 400 mW. Transepithelial PRK was performed using two different ablation protocols, to a total depth of 110 and 150  μm. The surface temperature was monitored during ablation; haze dynamics and histological samples were evaluated to assess outcomes of the PRK procedure. For comparison, analogous excimer ablation was performed. Increase of the ablation speed up to 1.6  s/diopter for a 6 mm optical zone using femtosecond UV pulses did not significantly impact the healing process.

A method for <italic<in vitro</italic< identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of <italic<Staphylococcus epidermidis</italic<. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs’ characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The <italic<in vitro</italic< model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial.

We present and discuss several modern optical methods based on elastic light scattering (ELS), along with their technical features and applications in biomedicine and life sciences. In particular, we review some ELS experiments at the single-cell level and explore new directions of applications. Due to recent developments in experimental systems (as shown in the literature), ELS lends itself to useful applications in the life sciences. Of the developed methods, we cover elastic scattering spectroscopy, optical tweezer-assisted measurement, goniometers, Fourier transform light scattering (FTLS), and microscopic methods. FTLS significantly extends the potential analysis of single cells by allowing monitoring of dynamical changes at the single-cell level. The main aim of our review is to demonstrate developments in the experimental investigation of ELS in single cells including issues related to theoretical “representations” and modeling of biological systems (cells, cellular systems, tissues, and so on). Goniometric measurements of ELS from optically trapped single cells are shown and the importance of the experimental verification of theoretical models of ELS in the context of biomedical applications is discussed.

Radiotherapy is one of the main methods to treat cancer. However, due to the propagation pattern of high-energy photons in tissue and their inability to discriminate between healthy and malignant tissues, healthy tissues may also be damaged, causing undesired side effects. A possible method for internal electron therapy, based on laser acceleration of electrons inside the patient’s body, is suggested. In this method, an optical waveguide, optimized for high intensities, is used to transmit the laser radiation and accelerate electrons toward the tumor. The radiation profile can be manipulated in order to create a patient-specific radiation treatment profile by changing the laser characteristics. The propagation pattern of electrons in tissues minimizes the side effects caused to healthy tissues. A simulation was developed to demonstrate the use of this method, calculating the trajectories of the accelerated electron as a function of laser properties. The simulation was validated by comparison to theory, showing a good fit for laser intensities of up to 2×10²⁰  (W/cm²), and was then used to calculate suggested treatment profiles for two tumor test cases (with and without penetration to the tumor). The results show that treatment profiles can be designed to cover tumor area with minimal damage to adjacent tissues.

An <italic<in vivo</italic< Raman spectroscopic study of amoeba (<italic<Acanthamoeba polyphaga</italic<) is presented. The changes of the spectra during the amoeba cyst activation and excystation are analyzed. The spectra show the changes of the relative intensities of bands corresponding to protein, lipid, and carotenoid components during cyst activation. The presence of carotenoids in the amoeba is observed via characteristic Raman bands. These signals in the Raman spectra are intense in cysts but decrease in intensity with cyst activation and exhibit a correlation with the life cycle of amoeba. This work demonstrates the feasibility of using Raman spectroscopy for the detection of single amoeba microorganisms <italic<in vivo</italic< and for the analysis of the amoeba life activity. The information obtained may have implications for the estimation of epidemiological situations and for the diagnostics and prognosis of the development of amoebic inflammations.

There are three possible mechanisms for 5-aminolevulinic acid (5-ALA) conjugated gold nanoparticles (GNPs) through electrostatic bonding for photodynamic therapy (PDT) of cancer: GNPs delivery function, singlet oxygen generation (SOG) by GNPs irradiated by light, and surface resonance enhancement (SRE) of SOG. Figuring out the exact mechanism is important for further clinical treatment. 5-ALA-GNPs and human chronic myeloid leukemia K562 cells were used to study delivery function and SOG by GNPs. The SRE of SOG enabled by GNPs was explored by protoporphyrin IX (PpIX)-GNPs conjugate through electrostatic bonding. Cell experiments show that the GNPs can improve the efficiency of PDT, which is due to the vehicle effect of GNPs. PpIX–GNPs conjugate experiments demonstrated that SOG can be improved about 2.5 times over PpIX alone. The experiments and theoretical results show that the local field enhancement (LFE) via localized surface plasmon resonance (LSPR) of GNPs is the major role; the LFE was dependent on the irradiation wavelength and the GNP’s size. The LFE increased with an increase of the GNP size (2R ≤50  nm). However, the LSPR function of the GNPs was not found in cell experiments. Our study shows that in 5-ALA-conjugated GNPs PDT, the delivery function of GNPs is the major role.

Traditionally, diffuse correlation spectroscopy (DCS) measures microvascular blood flow by fitting a physical model to the measurement of the intensity autocorrelation function from a single source-detector pair. This analysis relies on the accurate knowledge of the optical properties, absorption, and reduced scattering coefficients of the medium. Therefore, DCS is often deployed together with diffuse optical spectroscopy. We present an algorithm that employs multidistance DCS (MD-DCS) for simultaneous measurement of blood flow index, as well as an estimate of the optical properties of the tissue. The algorithm has been validated through noise-free and noise-added simulated data and phantom measurements. A longitudinal in vivo measurement of a mouse tumor is also shown. MD-DCS is introduced as a stand-alone system for small source-detector separations (<2  cm) for noninvasive measurement of microvascular blood flow.

Identification of tumor and normal cells is a promising application of Raman spectroscopy. The throughput of Raman-assisted cell sorting is limited by low sensitivity. Surface-enhanced Raman spectroscopy (SERS) is a well-recognized candidate to increase the intensity of Raman signals of cells. First, different strategies are summarized to detect tumor cells using targeted SERS probes. Then, a protocol is described to prepare multicore-SERS-labels (MSLs) by aggregating gold nanoparticles, coating with a reporter molecule and a thin silver shell to further boost enhancement, encapsulating with a stable silica layer, and functionalizing by epithelial cell adhesion molecule (EpCAM) antibodies. Raman, dark field and fluorescence microscopy proved the specific and nonspecific binding of functionalized and nonfunctionalized MSLs to MCF-7 tumor cells, leukocytes from blood, and nontransformed human foreskin fibroblasts. Raman imaging and dark field microscopy indicated no uptake of MSLs, yet binding to the cellular membrane. Viability tests were performed with living tumor cells to demonstrate the low toxicity of MSL-EpCAM. The SERS signatures were detected from cells with exposure times down to 25 ms at 785-nm laser excitation. The prospects of these MSLs in multiplex assays, for enumeration and sorting of circulating tumor cells in microfluidic chips, are discussed.

Tunable optical solitons generated by soliton self-frequency shift (SSFS) have become valuable tools for multiphoton microscopy (MPM). Recent progress in MPM using 1700 nm excitation enabled visualizing subcortical structures in mouse brain in vivo for the first time. Such an excitation source can be readily obtained by SSFS in a large effective-mode-area photonic crystal rod with a 1550-nm fiber femtosecond laser. A longpass filter was typically used to isolate the soliton from the residual in order to avoid excessive energy deposit on the sample, which ultimately leads to optical damage. However, since the soliton was not cleanly separated from the residual, the criterion for choosing the optimal filtering wavelength is lacking. Here, we propose maximizing the ratio between the multiphoton signal and the n’th power of the excitation pulse energy as a criterion for optimal spectral filtering in SSFS when the soliton shows dramatic overlapping with the residual. This optimization is based on the most efficient signal generation and entirely depends on physical quantities that can be easily measured experimentally. Its application to MPM may reduce tissue damage, while maintaining high signal levels for efficient deep penetration.

Fluorescence molecular tomography (FMT) as a noninvasive imaging modality has been widely used for biomedical preclinical applications. However, FMT reconstruction suffers from severe ill-posedness, especially when a limited number of projections are used. In order to improve the quality of FMT reconstruction results, a discrete cosine transform (DCT) based reweighted L1-norm regularization algorithm is proposed. In each iteration of the reconstruction process, different reweighted regularization parameters are adaptively assigned according to the values of DCT coefficients to suppress the reconstruction noise. In addition, the permission region of the reconstructed fluorophores is adaptively constructed to increase the convergence speed. In order to evaluate the performance of the proposed algorithm, physical phantom and <italic<in vivo</italic< mouse experiments with a limited number of projections are carried out. For comparison, different L1-norm regularization strategies are employed. By quantifying the signal-to-noise ratio (SNR) of the reconstruction results in the phantom and <italic<in vivo</italic< mouse experiments with four projections, the proposed DCT-based reweighted L1-norm regularization shows higher SNR than other L1-norm regularizations employed in this work.

The distribution of light intensity of three light-curing units (LCUs) to cure the resin-based composite for dental fillings was analyzed, and a homogeneity index [flat-top factor (FTF)] was calculated. The index is based on the M² index, which is used for laser beams. An optical spectrum analyzer was used with an optical fiber to produce an x-y power profile of each LCU light guide. The FTF-calculated values were 0.51 for LCU1 and 0.55 for LCU2, which was the best FTF, although it still differed greatly from the perfect FTF=1, and 0.27 for LCU3, which was the poorest value and even lower than the Gaussian FTF=0.5. All LCUs presented notably heterogeneous light distribution, which can lead professionals and researchers to produce samples with irregular polymerization and poor mechanical properties.

The aim of this study was to evaluate the surface roughness and wettability of dentin following ultrashort pulsed laser ablation with different levels of fluence and pulse overlap (PO). Twenty-five extracted human teeth crowns were cut longitudinally into slices of approximately 1.5-mm thick and randomly divided into nine groups of five. Samples in groups 1 to 8 were ablated with an ultrashort pulsed laser through a galvanometric scanning system. Samples in group 9 were prepared using a mechanical rotary instrument. The surface roughness of samples from each group was then measured using a three-dimensional profile measurement laser microscope, and wettability was evaluated by measuring the contact angle of a drop of water on the prepared dentin surface using an optical contact angle measuring device. The results showed that both laser fluence and PO had an effect on dentin surface roughness. Specifically, a higher PO decreased dentin surface roughness and reduced the effect of high-laser fluence on decreasing the surface roughness in some groups. Furthermore, all ablated dentin showed a contact angle of approximately 0 deg, meaning that laser ablation significantly improved wettability. Adjustment of ultrashort pulsed laser parameters can, therefore, significantly alter dentin surface roughness and wettability.

An interferometer for measuring dynamic properties of the in vivo tear film on the human cornea has been developed. The system is a near-infrared instantaneous phase-shifting Twyman-Green interferometer. The laser source is a 785 nm solid-state laser, and the system has been carefully designed and calibrated to ensure that the system operates at eye-safe levels. Measurements are made over a 6 mm diameter on the cornea. Successive frames of interferometric height measurements are combined to produce movies showing both the quantitative and qualitative changes in the topography of the tear film surface and structure. To date, measurement periods of up to 120 s at 28.6 frames per second have been obtained. Several human subjects have been examined using this system, demonstrating a surface height resolution of 25 nm and spatial resolution of 6  μm. Examples of features that have been observed in these preliminary studies of the tear film include postblink disruption, evolution, and stabilization of the tear film; tear film artifacts generated by blinking; tear film evaporation and breakup; and the propagation of foreign objects in the tear film. This paper discusses the interferometer design and presents results from in vivo measurements.

To simplify imaging focusing and calibration tasks, a laser-scanning microscope needs to scan at a moderate frame rate. The inertia of a galvanometric scanner leads to time delays when following external commands, which subsequently introduces image distortions that deteriorate as scan frequency increases. Sinusoidal and triangular waveforms were examined as fast axis driving patterns. The interplay among driving pattern, frequency, sampling rate, phase shift, linear scanning range, and their effect on reconstructed images was discussed. Utilizing position feedback from the linear galvo scanners, the effect of response time could be automatically compensated in real time. Precompensated triangular driving waveform offered the least amount of image distortion.

Current imaging tools are associated with inconsistent sensitivity and specificity for detection of Barrett’s-associated neoplasia. Optical imaging has shown promise in improving the classification of neoplasia in vivo. The goal of this pilot study was to evaluate whether in vivo vital dye fluorescence imaging (VFI) has the potential to improve the accuracy of early-detection of Barrett’s-associated neoplasia. In vivo endoscopic VFI images were collected from 65 sites in 14 patients with confirmed Barrett’s esophagus (BE), dysplasia, or esophageal adenocarcinoma using a modular video endoscope and a high-resolution microendoscope (HRME). Qualitative image features were compared to histology; VFI and HRME images show changes in glandular structure associated with neoplastic progression. Quantitative image features in VFI images were identified for objective image classification of metaplasia and neoplasia, and a diagnostic algorithm was developed using leave-one-out cross validation. Three image features extracted from VFI images were used to classify tissue as neoplastic or not with a sensitivity of 87.8% and a specificity of 77.6% (AUC=0.878). A multimodal approach incorporating VFI and HRME imaging can delineate epithelial changes present in Barrett’s-associated neoplasia. Quantitative analysis of VFI images may provide a means for objective interpretation of BE during surveillance.

Of the three measurement schemes established for diffuse fluorescence tomography (DFT), the time-domain scheme is well known to provide the richest information about the distribution of the targeting fluorophore in living tissues. However, the explicit use of the full time-resolved data usually leads to a considerably lengthy time for image reconstruction, limiting its applications to three-dimensional or small-volume imaging. To cope with the adversity, we propose herein a computationally efficient scheme for DFT image reconstruction where the time-dependent photon density is expanded to a Fourier-series and calculated by solving the independent frequency-domain diffusion equations at multiple sampling frequencies with the support of a combined multicore CPU-based coarse-grain and multithread GPU-based fine-grain parallelization strategy. With such a parallelized Fourier-series truncated diffusion approximation, both the time- and frequency-domain inversion procedures are developed and validated for their effectiveness and accuracy using simulative and phantom experiments. The results show that the proposed method can generate reconstructions comparable to the explicit time-domain scheme, with significantly reduced computational time.

Photoacoustic imaging can achieve high-resolution three-dimensional (3-D) visualization of optical absorbers at penetration depths of ∼1  cm in biological tissues by detecting optically induced high ultrasound frequencies. Tomographic acquisition with ultrasound linear arrays offers an easy implementation of single-side access, parallelized, and high-frequency detection, but usually comes with an image quality impaired by the directionality of the detectors. Indeed, a simple translation of the array perpendicular to its median imaging plane is often used, but results both in a poor resolution in the translation direction and strong limited-view artifacts. To improve the spatial resolution and the visibility of complex structures while retaining a planar detection geometry, we introduce, in this paper, a rotate-translate scanning scheme and investigate the performance of a scanner implemented at 15 MHz center frequency. The developed system achieved a quasi-isotropic uniform 3-D resolution of ∼170  μm over a cubic volume of side length 8.5 mm, i.e., an improvement in the resolution in the translation direction by almost one order of magnitude. Dual-wavelength imaging was also demonstrated with ultrafast wavelength shifting. The validity of our approach was shown in vitro. We discuss the ability to enable in vivo imaging for preclinical and clinical studies.

An increasing number of integrated optical and acoustic intravascular imaging systems have been developed and hold great promise for accurately diagnosing vulnerable plaques and guiding atherosclerosis treatment. However, in any intravascular environment, the vascular lumen is filled with blood, a high-scattering source for optical and high-frequency ultrasound signals. Blood must be flushed away to provide clearer images. To our knowledge, no research has been performed to find the ideal flushing agent for combined optical and acoustic imaging techniques. We selected three solutions as potential flushing agents for their image-enhancing effects: mannitol, dextran, and iohexol. Testing of these flushing agents was performed in a closed-loop circulation model and <italic<in vivo</italic< on rabbits. We found that a high concentration of dextran was the most useful for simultaneous intravascular ultrasound and optical coherence tomography imaging.

The measurement of central corneal thickness (CCT) is important in ophthalmology. Most studies concerned the value at normal status, while rare ones focused on its dynamic changing. The commercial Corvis ST is the only commercial device currently available to visualize the two-dimensional image of dynamic corneal profiles during an air puff indentation. However, the directly observed CCT involves the Scheimpflug distortion, thus misleading the clinical diagnosis. This study aimed to correct the distortion for better measuring the dynamic CCTs. The optical path was first derived to consider the influence of factors on the use of Covis ST. A correction method was then proposed to estimate the CCT at any time during air puff indentation. Simulation results demonstrated the feasibility of the intuitive-feasible calibration for measuring the stationary CCT and indicated the necessity of correction when air puffed. Experiments on three contact lenses and four human corneas verified the prediction that the CCT would be underestimated when the improper calibration was conducted for air and overestimated when it was conducted on contact lenses made of polymethylmethacrylate. Using the proposed method, the CCT was finally observed to increase by 66±34  μm at highest concavity in 48 normal human corneas.

We describe rare-earth-doped nanophosphors (RE-NPs) for biological imaging using cathodoluminescence (CL) microscopy based on scanning transmission electron microscopy (STEM). We report the first demonstration of multicolor CL nanobioimaging using STEM with nanophosphors. The CL spectra of the synthesized nanophosphors (Y₂O₃:Eu, Y₂O₃:Tb) were sufficiently narrow to be distinguished. From CL images of RE-NPs on an elastic carbon-coated copper grid, the spatial resolution was beyond the diffraction limit of light. Y₂O₃:Tb and Y₂O₃:Eu RE-NPs showed a remarkable resistance against electron beam exposure even at high acceleration voltage (80 kV) and retained a CL intensity of more than 97% compared with the initial intensity for 1 min. In biological CL imaging with STEM, heavy-metal-stained cell sections containing the RE-NPs were prepared, and both the CL images of RE-NPs and cellular structures, such as mitochondria, were clearly observed from STEM images with high contrast. The cellular CL imaging using RE-NPs also had high spatial resolution even though heavy-metal-stained cells are normally regarded as highly scattering media. Moreover, since the RE-NPs exhibit photoluminescence (PL) excited by UV light, they are useful for multimodal correlative imaging using CL and PL.

Noninvasive middle ear imaging using optical coherence tomography (OCT) presents some unique challenges for real-time, clinical use in humans. We present results from a two-dimensional/three-dimensional OCT system built to assess the imaging requirements of clinical middle ear imaging, and the technical challenges associated with them. These include the need to work at a low numerical aperture, the deleterious effects of transtympanic imaging on image quality at the ossicles, sensitivity requirements for clinical fidelity of images at real-time rates, and the high dynamic-range requirements of the ear. We validated the system by imaging cadaveric specimens with simulated disorders to show the clinical applicability of the images. We also provide additional insight into the likely role of OCT in clinical otology.

An optimized approach to nonlinear iterative reconstruction of magnetic resonance imaging (MRI)–guided near-infrared spectral tomography (NIRST) images was developed using an L-curve-based algorithm for the choice of regularization parameter. This approach was applied to clinical exam data to maximize the reconstructed values differentiating malignant and benign lesions. MRI/NIRST data from 25 patients with abnormal breast readings (BI-RADS category 4-5) were analyzed using this optimal regularization methodology, and the results showed enhanced p values and area under the curve (AUC) for the task of differentiating malignant from benign lesions. Of the four absorption parameters and two scatter parameters, the most significant differences for benign versus malignant were total hemoglobin (HbT) and tissue optical index (TOI) with pvalues=0.01 and 0.001, and AUC values=0.79 and 0.94, respectively, in terms of HbT and TOI. This dramatically improved the values relative to fixed regularization (pvalue=0.02 and 0.003; AUC=0.75 and 0.83) showing that more differentiation was possible with the optimal method. Through a combination of both biomarkers, HbT and TOI, the AUC increased from 82.9% (fixed regulation=0.1) to 94.3% (optimal method).

Light reflectance and transmission from soft tissue has been utilized in noninvasive clinical measurement devices such as the photoplethysmograph (PPG) and reflectance pulse oximeter. Incident light on the skin travels into the underlying layers and is in part reflected back to the surface, in part transferred and in part absorbed. Most methods of near infrared (NIR) spectroscopy focus on the volume reflectance from a semi-infinite sample, while very few measure transmission. We have previously shown that examining the full scattering profile (angular distribution of exiting photons) provides more comprehensive information when measuring from a cylindrical tissue. Furthermore, an isobaric point was found which is not dependent on changes in the reduced scattering coefficient. The angle corresponding to this isobaric point depends on the tissue diameter. We investigated the role of multiple scattering and absorption on the full scattering profile of a cylindrical tissue. First, we define the range in which multiple scattering occurs for different tissue diameters. Next, we examine the role of the absorption coefficient in the attenuation of the full scattering profile. We demonstrate that the absorption linearly influences the intensity at each angle of the full scattering profile and, more importantly, the absorption does not change the position of the isobaric point. The findings of this work demonstrate a realistic model for optical tissue measurements such as NIR spectroscopy, PPG, and pulse oximetery.

Functional near-infrared spectroscopy is prone to contamination by motion artifacts (MAs). Motion correction algorithms have previously been proposed and their respective performance compared for evoked brain activation studies. We study instead the effect of MAs on “oscillation” data which is at the basis of functional connectivity and autoregulation studies. We use as our metric of interest the interhemispheric correlation (IHC), the correlation coefficient between symmetrical time series of oxyhemoglobin oscillations. We show that increased motion content results in a decreased IHC. Using a set of motion-free data on which we add real MAs, we find that the best motion correction approach consists of discarding the segments of MAs following a careful approach to minimize the contamination due to band-pass filtering of data from “bad” segments spreading into adjacent “good” segments. Finally, we compare the IHC in a stroke group and in a healthy group that we artificially contaminated with the MA content of the stroke group, in order to avoid the confounding effect of increased motion incidence in the stroke patients. After motion correction, the IHC remains lower in the stroke group in the frequency band around 0.1 and 0.04 Hz, suggesting a physiological origin for the difference. We emphasize the importance of considering MAs as a confounding factor in oscillation-based functional near-infrared spectroscopy studies.

Mueller matrix polarimetry along with polar decomposition algorithm was employed for the characterization of ex vivo normal and adenocarcinoma human colon tissues by polarized light in the visible spectral range (425–725 nm). Six derived polarization metrics [total diattenuation (D_T), retardance (R_T), depolarization (Δ_T), linear diattenuation (D_L), retardance (δ), and depolarization (Δ_L)] were compared for normal and adenocarcinoma colon tissue samples. The results show that all six polarimetric properties for adenocarcinoma samples were significantly higher as compared to the normal samples for all wavelengths. The Wilcoxon rank sum test illustrated that total retardance is a good candidate for the discrimination of normal and adenocarcinoma colon samples. Support vector machine classification for normal and adenocarcinoma based on the four polarization properties spectra (Δ_T, Δ_L, R_T,and δ) yielded 100% accuracy, sensitivity, and specificity, while both D_T and D_L  showed 66.6%, 33.3%, and 83.3% accuracy, sensitivity, and specificity, respectively. The combination of polarization analysis and given classification methods provides a framework to distinguish the normal and cancerous tissues.

We developed multimodal multiphoton microspectroscopy using a small-diameter probe with gradient-index lenses and applied it to unstained Alzheimer’s disease (AD) brain samples. Our system maintained the image quality and spatial resolution of images obtained using an objective lens of similar numerical aperture. Multicolor images of AD brain samples were obtained simultaneously by integrating two-photon excited fluorescence and second-harmonic generation on a coherent anti-Stokes Raman scattering (CARS) microendoscope platform. Measurements of two hippocampal regions, the cornus ammonis-1 and dentate gyrus, revealed more lipids, amyloid fibers, and collagen in the AD samples than in the normal samples. Normal and AD brains were clearly distinguished by a large spectral difference and quantitative analysis of the CH mode using CARS microendoscope spectroscopy. We expect this system to be an important diagnosis tool in AD research.

A noninvasive optical technique, which is based on a combination of reflectance spectroscopy and gas in scattering media absorption spectroscopy, is demonstrated. It has the potential to improve diagnostics of middle ear infections. An ear phantom prepared with a tissue cavity, which was covered with scattering material, was used for spectroscopic measurements. Diffuse reflectance spectra of the phantom eardrum were measured with a reflectance probe. The presence of oxygen and water vapor as well as gas exchange in the phantom cavity were studied with a specially designed fiber-optic probe for backscattering detection geometry. The results suggest that this method can be developed for improved clinical detection of middle ear infection.

Recently, a multidiameter single-fiber reflectance and fluorescence spectroscopy device has been developed that enabled us to extract the autofluorescence of tissue that is corrected for the optical properties. Such a system has been incorporated in the population-based Rotterdam Study to investigate the autofluorescence of the skin. Since the device will be used by different operators over many years, it is essential that the results are comparable between users. It is, however, unclear how different methods of handling the probe might influence the outcome. Variability of blood oxygen saturation, blood volume fraction and vessel diameter, average gamma, reduced scattering coefficient at 800 nm, and integrated intrinsic fluorescence measured in three volunteers were assessed within and between eight untrained users. A variability of less than one standard deviation from the group mean was defined as an acceptable limit. Three mature volunteers were also included to assess the intrauser variability of mature skin. The variation in the measured parameters suggests that variation is dominated by tissue heterogeneity. Most users measured within one standard deviation of the group mean. Notably, corrected intrinsic fluorescence showed low intra- and interuser variability. These results strongly suggest that variability is mostly caused by tissue heterogeneity and is not user induced.

The work is devoted to the study of sizes and concentrations of proteins, and their aggregates in blood plasma samples, using static and dynamic light scattering methods. A new approach is proposed based on multiple repetition of measurements of intensity size distribution and on counting the number of registrations of different sizes, which made it possible to obtain statistically confident particle sizes and concentrations in the blood plasma. It was revealed that statistically confident particle sizes in the blood plasma were stable during 30 h of observations, whereas the concentrations of particles of different sizes varied as a result of redistribution of material between them owing to the protein degradation processes.

Liquid crystal (LC)-based biosensing has attracted much attention in recent years. We focus on improving the detection limit of LC-based immunoassay techniques by surface modification of the surfactant alignment layer consisting of dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP). The cancer biomarker CA125 was detected with an array of anti-CA125 antibodies immobilized on the ultraviolet (UV)-modified DMOAP monolayer. Compared with a pristine counterpart, UV irradiation enhanced the binding affinity of the CA125 antibody and reproducibility of immunodetection in which a detection limit of 0.01  ng/ml for the cancer biomarker CA125 was achieved. Additionally, the optical texture observed under a crossed polarized microscope was correlated with the analyte concentration. In a proof-of-concept experiment using CA125-spiked human serum as the analyte, specific binding between the CA125 antigen and the anti-CA125 antibody resulted in a distinct and concentration-dependent optical response despite the high background caused by nonspecific binding of other biomolecules in the human serum. Results from this study indicate that UV modification of the alignment layer, as well as detection with LCs of large birefringence, contributes to the enhanced performance of the label-free LC-based immunodetection, which may be considered a promising alternative to conventional label-based methods.

The present study reports a noninvasive technique for the measurement of the pulse transit time differential (PTTD) from the pulse pressure waveforms obtained at the carotid artery and radial artery using fiber Bragg grating pulse recorders (FBGPR). PTTD is defined as the time difference between the arrivals of a pulse pressure waveform at the carotid and radial arterial sites. The PTTD is investigated as an indicator of variation in the systolic blood pressure. The results are validated against blood pressure variation obtained from a Mindray Patient Monitor. Furthermore, the pulse wave velocity computed from the obtained PTTD is compared with the pulse wave velocity obtained from the color Doppler ultrasound system and is found to be in good agreement. The major advantage of the PTTD measurement via FBGPRs is that the data acquisition system employed can simultaneously acquire pulse pressure waveforms from both FBGPRs placed at carotid and radial arterial sites with a single time scale, which eliminates time synchronization complexity.

Oxidative stress has become an exciting area of schizophrenia (SCZ) research, and provides ample opportunities and hope for a better understanding of its pathophysiology, which may lead to new treatment strategies. The first objective of the present study was to analyze the oxidative stress markers in breath samples of patients with SCZ before and after the treatment with Levomepromazine. The second objective was to analyze the deficiency of amino acids marker in breath samples of patients with SCZ before and after the treatment. Exhaled breath was collected from 15 SCZ patients and 19 healthy controls; subsequently, CO₂ laser photoacoustic spectroscopy was used to assess the exhaled breath compounds of the study subjects. One of the main breath biomarkers of the oxidative stress is ethylene, while one of the main breath biomarkers of the amino acids deficiency is ammonia. The breath biomarkers in the exhalation of SCZ patients exhibited significant differences from the breath biomarkers in the exhalation of healthy controls. Analysis of breath ethylene and breath ammonia provides a related model of SCZ exhalation that could represent an effective and convenient screening method for this intellectual disability.

Photodynamic therapy (PDT) is an emerging treatment modality for various diseases, especially for cancer therapy. Although high efficacy is demonstrated for PDT using standardized protocols in nonhyperkeratotic actinic keratoses, alternative light doses expected to increase efficiency, to reduce adverse effects or to expand the use of PDT, are still being evaluated and refined. We propose a comparison of the three most common light doses in the treatment of actinic keratosis with 5-aminolevulinic acid PDT through mathematical modeling. The proposed model is based on an iterative procedure that involves determination of the local fluence rate, updating of the local optical properties, and estimation of the local damage induced by the therapy. This model was applied on a simplified skin sample model including an actinic keratosis lesion, with three different light doses (red light dose, 37  J/cm², 75  mW/cm², 500 s; blue light dose, 10  J/cm², 10  mW/cm², 1000 s; and daylight dose, 9000 s). Results analysis shows that the three studied light doses, although all efficient, lead to variable local damage. Defining reference damage enables the nonoptimal parameters for the current light doses to be refined and the treatment to be more suitable.

This PDF file contains the errata for “JBO Vol. 20 Issue 05 Paper JBO-2015-0217-ERR” for JBO Vol. 20 Issue 05

This PDF file contains the errata for “JBO Vol. 20 Issue 05 Paper JBO-2015-0515-ERR” for JBO Vol. 20 Issue 05