Photoacoustic imaging (PA imaging or PAI) has shown great promise in the detection and monitoring of cancer. Although nanocarrier-based contrast agents have been studied for use in PAI, small molecule contrast agents are required due to their ease of preparation, cost-effectiveness, and low toxicity. Here, we evaluated the usefulness of a novel cyanine dye IC7-1-Bu as a PAI contrast agent without conjugated targeting moieties for in vivo tumor imaging in a mice model. Basic PA characteristics of IC7-1-Bu were compared with indocyanine green (ICG), a Food and Drug Administration approved dye, in an aqueous solution. We evaluated the tumor accumulation profile of IC7-1-Bu and ICG by in vivo fluorescence imaging. In vivo PAI was then performed with a photoacoustic tomography system 24 and 48 h after intravenous injection of IC7-1-Bu into tumor bearing mice. IC7-1-Bu showed about a 2.3-fold higher PA signal in aqueous solution compared with that of ICG. Unlike ICG, IC7-1-Bu showed high tumor fluorescence after intravenous injection. In vivo PAI provided a tumor to background PA signal ratio of approximately 2.5 after intravenous injection of IC7-1-Bu. These results indicate that IC7-1-Bu is a promising PAI contrast agent for cancer imaging without conjugation of targeting moieties.

We report on a phase-based method for accurately measuring the ocular pulse in the anterior chamber in vivo. Using phase-sensitive optical coherence tomography with optimized scanning protocols and equations for compensating bulk motion and environmental vibrations, a high sensitivity of 0.9  μm/s minimal velocity is demonstrated at a wide detection band of 0 to 380 Hz. The pulsatile relative motion between cornea and crystalline lens in rodents is visualized and quantified. The relative motion is most likely caused by respiration (1.6 Hz) and heartbeat (6.6 Hz). The velocity amplitude of the relative motion is 10.3±2.4  μm/s . The displacement amplitudes at the respiratory and cardiac frequencies are 202.5±64.9 and 179.9±49.4  nm , respectively. The potential applications of the measurement technique can be found in the evaluation of intraocular pressure and the measurement of biomechanical properties of the ocular tissue, which are important in several ocular diseases.

In breast-conserving surgery, an optical wire is a useful surgical guiding tool to optically locate small lesions within the breast tissue. However, the use of a long silica glass fiber as the optical wire can be burdensome to patients because of its stiffness and nonbiocompatibility. We investigate the use of a biocompatible fiber for light localization in tissue. A surgical suture with a diameter of 400  μm and a few centimeters long is employed as the biocompatible optical waveguide to transport the visible laser light to the inner tissue site. Optical location is confirmed with glow ball-like red laser illumination at the tip of the suture embedded within a fresh chicken breast tissue. Effective optical power coupling to the suture is made by using a double-cladding fiber coupler. From this preliminary result, we realize practical light localization with biopolymer waveguides.

Contrast agents have shown to be useful in the detection of cancers. The goal of this study was to compare enhancement of brain cancer contrast using reflectance and fluorescence confocal imaging of two fluorophores, methylene blue (MB) and demeclocycline (DMN). MB absorbs light in the red spectral range and fluoresces in the near-infrared. It is safe for in vivo staining of human skin and breast tissue. However, its safety for staining human brain is questionable. Thus, DMN, which absorbs light in the violet spectral range and fluoresces between 470 and 570 nm, could provide a safer alternative to MB. Fresh human gliomas, obtained from surgeries, were cut in half and stained with aqueous solutions of MB and DMN, respectively. Stained tissues were imaged using multimodal confocal microscopy. Resulting reflectance and fluorescence optical images were compared with hematoxylin and eosin histopathology, processed from each imaged tissue. Results indicate that images of tissues stained with either stain exhibit comparable contrast and resolution of morphological detail. Further studies are required to establish the safety and efficacy of these contrast agents for use in human brain.

We introduce a technique that limits absorption effects in fluorescence imaging and does not require extensive imaging processing, thus allowing for video rate imaging. The absorption minimization is achieved using spatial frequency domain imaging at a single high spatial frequency with standard three-phase demodulation. At a spatial frequency f=0.5  mm⁻¹, we demonstrated in both in-vitro phantoms and ex-vivo tissue that the absorption can be significantly reduced. In the real-time implementation, we achieved a video rate of 19  frames/s. This technique has potential in cancer visualization and tumor margin detection.

Knowledge of neuronal wiring and morphogenesis in Drosophila is essential to understand brain function and dysfunction. The immunoenzyme method based on horseradish peroxidase/diaminobenzidine (HRP/DAB) provides high-contrast images to resolve details underlying neuronal architecture. However, the poor staining penetration and a lack of corresponding three-dimensional imaging methodology limit its application. Herein, we modified the HRP/DAB method to stain neuronal circuits in the whole brain of Drosophila. Furthermore, we found that imaging with the micro-optical sectioning tomography system provided a fast and automatic method that could dissect cell-specific neuroanatomical architecture at a submicron voxel resolution.

Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques.

Multiphoton microscopy is the preferred method when in vivo deep-tissue imaging is required. This review presents the application of multiphoton microscopy in defining liver function. In particular, multiphoton microscopy is useful in imaging intracellular events, such as mitochondrial depolarization and cellular metabolism in terms of NAD(P)H changes with fluorescence lifetime imaging microscopy. The morphology of hepatocytes can be visualized without exogenously administered fluorescent dyes by utilizing their autofluorescence and second harmonic generation signal of collagen, which is useful in diagnosing liver disease. More specific imaging, such as studying drug transport in normal and diseased livers are achievable, but require exogenously administered fluorescent dyes. If these techniques can be translated into clinical use to assess liver function, it would greatly improve early diagnosis of organ viability, fibrosis, and cancer.

We obtained backscattering Mueller matrix images on a cancerous tissue sample through a gradient-index (GRIN) lens and observed strong distortion in all the Mueller matrix elements. By measuring the intrinsic polarization properties of the GRIN lens, which is dominated by birefringence following a radial profile, and applying a matrix inversion method to the distorted Mueller matrix, we are able to remove the artifacts because of the birefringent GRIN lens and recover the polarization features of the sample. The results demonstrate the feasibility to take Mueller matrix measurements using GRIN lenses or other optical components with strong birefringence.

The color of an object is perceived differently depending on the ambient light conditions. Since dental all-ceramic restorations are fabricated by building up several layers to reproduce the tooth shade, the optical properties of each layer should be optimized for successful shade reproduction. This study aimed to determine the separate contributions of the color shifts in each of the core and veneer layers of all-ceramics by switching the illuminating lights on the color shifts of layered ceramics. Specimens of seven kinds of core ceramics and the corresponding veneer ceramics for each core were fabricated with a layered thickness of 1.5 mm. A sintering ceramic was used as a reference core material. The Commission Internationale de l’Eclairage (CIE) color coordinates of core, veneer, and layered specimens were measured with a spectroradiometer under the CIE illuminant D65 (daylight), A (incandescent lamp), and F9 (fluorescent lamp) simulating lights. Color shifts of the layered specimens were primarily determined by the CIE a* shifts (D65 to A switch) or by the CIE b* shifts (D65 to F9 switch) of the veneer layer. The color coordinates shifts in the constituent layers differentially influenced those of the layered specimens by the kind of switched lights. Therefore, the optical properties of the constituent layers of all-ceramics should be controlled to reflect these findings.

Understanding the human hearing process would be helped by quantification of the transient mechanical response of the human ear, including the human tympanic membrane (TM or eardrum). We propose a new hybrid high-speed holographic system (HHS) for acquisition and quantification of the full-field nanometer transient (i.e., <10  kHz ) displacement of the human TM. We have optimized and implemented a 2+1 frame local correlation (LC) based phase sampling method in combination with a high-speed (i.e., <;40  K fps ) camera acquisition system. To our knowledge, there is currently no existing system that provides such capabilities for the study of the human TM. The LC sampling method has a displacement difference of <11  nm relative to measurements obtained by a four-phase step algorithm. Comparisons between our high-speed acquisition system and a laser Doppler vibrometer indicate differences of <10  μs . The high temporal (i.e., <40  kHz ) and spatial (i.e., <100  k data points) resolution of our HHS enables parallel measurements of all points on the surface of the TM, which allows quantification of spatially dependent motion parameters, such as modal frequencies and acoustic delays. Such capabilities could allow inferring local material properties across the surface of the TM.

Photoacoustic (PA) imaging has emerged as a noninvasive diagnostic method which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated with laser. For tumor diagnosis, PA contrast agent has been proposed to enhance the PA effect for detecting tumors sensitively. Here, we prepared a human serum albumin (HSA) conjugated with indocyanine green (ICG) as a PA contrast agent allowing enhanced permeability and retention effect for sensitive tumor imaging. The feasibility of PA imaging with HSA-ICG to detect allografted tumors was evaluated in tumor-bearing mice. In vivo fluorescence imaging and radiolabeled biodistribution study showed that the biodistribution dramatically changed as the number of ICG bound to HSA increased, and the maximum accumulation of ICG was achieved when around three ICG molecules were loaded on an HSA. In vivo PA imaging demonstrated a tumor-selective and dose-dependent increase of PA signal intensity in mice injected with HSA-ICG (R ² =0.88 , 387% increase for HSA-ICG, 104 nmol ICG). In conclusion, HSA-ICG clearly visualized the allografted tumors with high tumor-to-background ratios having high quantitative and spatial resolution for the sensitive PA imaging of tumors. HSA-ICG could be useful as a favorable contrast agent for PA tumor imaging for the management of cancer.

We present an application of spatial frequency-domain imaging (SFDI) to the wide-field imaging of drug delivery to brain tissue. Measurements were compared with values obtained by a previously validated variation of diffuse reflectance spectroscopy, the method of optical pharmacokinetics (OP). We demonstrate a cross-correlation between the two methods for absorption extraction and drug concentration determination in both experimental tissue phantoms and freshly extracted rodent brain tissue. These methods were first used to assess intra-arterial (IA) delivery of cationic liposomes to brain tissue in Sprague Dawley rats under transient cerebral hypoperfusion. Results were found to be in agreement with previously published experimental data and pharmacokinetic models of IA drug delivery. We then applied the same scheme to evaluate IA mitoxantrone delivery to glioma-bearing rats. Good correlation was seen between OP and SFDI determined concentrations taken from normal and tumor averaged sites. This study shows the feasibility of mapping drug/tracer distributions and encourages the use of SFDI for spatial imaging of tissues for drug/tracer-tagged carrier deposition and pharmacokinetic studies.

Quantitatively reconstructing optical absorption using photoacoustic imaging is nontrivial. Theoretical hurdles, such as nonuniqueness and numerical instability, can be mitigated by using multiple illuminations. However, even with multiple illuminations, using ANSI-safety-limited fluence for practical imaging may result in poor performance owing to limited signal-to-noise ratio (SNR). We demonstrate the use of S-sequence coded patterned illumination to boost SNR while preserving the enhanced stability of multiple-illumination iterative techniques.

Fluorescence-guided diagnosis of tumor tissue is in many cases insufficient, because false positive results interfere with the outcome. Improvement through observation of cell metabolism might offer the solution, but needs a detailed understanding of the origin of autofluorescence. With respect to this, spectrally resolved multiphoton fluorescence lifetime imaging was investigated to analyze cell metabolism in metabolic phenotypes of malignant and nonmalignant oral mucosa cells. The time-resolved fluorescence characteristics of NADH were measured in cells of different origins. The fluorescence lifetime of bound and free NADH was calculated from biexponential fitting of the fluorescence intensity decay within different spectral regions. The mean lifetime was increased from nonmalignant oral mucosa cells to different squamous carcinoma cells, where the most aggressive cells showed the longest lifetime. In correlation with reports in the literature, the total amount of NADH seemed to be less for the carcinoma cells and the ratio of free/bound NADH was decreased from nonmalignant to squamous carcinoma cells. Moreover for squamous carcinoma cells a high concentration of bound NADH was found in cytoplasmic organelles (mainly mitochondria). This all together indicates that oxidative phosphorylation and a high redox potential play an important role in the energy metabolism of these cells.

Functional near-infrared spectroscopy (fNIRS) is an optical method for noninvasively determining brain activation by estimating changes in the absorption of near-infrared light. Diffuse optical tomography (DOT) extends fNIRS by applying overlapping “high density” measurements, and thus providing a three-dimensional imaging with an improved spatial resolution. Reconstructing brain activation images with DOT requires solving an underdetermined inverse problem with far more unknowns in the volume than in the surface measurements. All methods of solving this type of inverse problem rely on regularization and the choice of corresponding regularization or convergence criteria. While several regularization methods are available, it is unclear how well suited they are for cerebral functional DOT in a semi-infinite geometry. Furthermore, the regularization parameter is often chosen without an independent evaluation, and it may be tempting to choose the solution that matches a hypothesis and rejects the other. In this simulation study, we start out by demonstrating how the quality of cerebral DOT reconstructions is altered with the choice of the regularization parameter for different methods. To independently select the regularization parameter, we propose a cross-validation procedure which achieves a reconstruction quality close to the optimum. Additionally, we compare the outcome of seven different image reconstruction methods for cerebral functional DOT. The methods selected include reconstruction procedures that are already widely used for cerebral DOT [minimum ℓ₂-norm estimate (ℓ₂MNE) and truncated singular value decomposition], recently proposed sparse reconstruction algorithms [minimum ℓ1- and a smooth minimum ℓ0-norm estimate (ℓ₁MNE, ℓ₀MNE, respectively)] and a depth- and noise-weighted minimum norm (wMNE). Furthermore, we expand the range of algorithms for DOT by adapting two EEG-source localization algorithms [sparse basis field expansions and linearly constrained minimum variance (LCMV) beamforming]. Independent of the applied noise level, we find that the LCMV beamformer is best for single spot activations with perfect location and focality of the results, whereas the minimum ℓ1-norm estimate succeeds with multiple targets.

Remodeling of the extracellular matrix has been implicated in ovarian cancer. To quantitate the remodeling, we implement a form of texture analysis to delineate the collagen fibrillar morphology observed in second harmonic generation microscopy images of human normal and high grade malignant ovarian tissues. In the learning stage, a dictionary of “textons”—frequently occurring texture features that are identified by measuring the image response to a filter bank of various shapes, sizes, and orientations—is created. By calculating a representative model based on the texton distribution for each tissue type using a training set of respective second harmonic generation images, we then perform classification between images of normal and high grade malignant ovarian tissues. By optimizing the number of textons and nearest neighbors, we achieved classification accuracy up to 97% based on the area under receiver operating characteristic curves (true positives versus false positives). The local analysis algorithm is a more general method to probe rapidly changing fibrillar morphologies than global analyses such as FFT. It is also more versatile than other texture approaches as the filter bank can be highly tailored to specific applications (e.g., different disease states) by creating customized libraries based on common image features.

Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear–cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

We adapt a graphics processing unit (GPU) to dynamic quantitative second-harmonic generation imaging. We demonstrate the temporal advantage of the GPU-based approach by computing the number of frames analyzed per second from SHG image videos showing varying fiber orientations. In comparison to our previously reported CPU-based approach, our GPU-based image analysis results in ∼10× improvement in computational time. This work can be adapted to other quantitative, nonlinear imaging techniques and provides a significant step toward obtaining quantitative information from fast in vivo biological processes.

The healthy adult human retina contains in its macular region a high concentration of blue-light absorbing carotenoid compounds, known as macular pigment (MP). Consisting of the carotenoids lutein, zeaxanthin, and meso-zeaxanthin, the MP is thought to shield the vulnerable tissue layers in the retina from light-induced damage through its function as an optical attenuator and to protect the tissue cells within its immediate vicinity through its function as a potent antioxidant. Autofluorescence imaging (AFI) is emerging as a viable optical method for MP screening of large subject populations, for tracking of MP changes over time, and for monitoring MP uptake in response to dietary supplementation. To investigate the influence of ocular media opacities on AFI-based MP measurements, in particular, the influence of lens cataracts, we conducted a clinical trial with a large subject population (93 subjects) measured before and after cataract surgery. General AFI image contrast, retinal blood vessel contrast, and presurgery lens opacity scores [Lens Opacities Classification System III (LOCS III)] were investigated as potential predictors for image degradation. These clinical results show that lens cataracts can severely degrade the achievable pixel contrasts in the AFI images, which results in nominal MP optical density levels that are artifactually reduced. While LOCS III scores and blood vessel contrast are found to be only a weak predictor for this effect, a strong correlation exists between the reduction factor and the image contrast, which can be quantified via pixel intensity histogram parameters. Choosing the base width of the histogram, the presence or absence of ocular media opacities can be determined and, if needed, the nominal MP levels can be corrected with factors depending on the strength of the opacity.

Accurate quantification of microvasculature remains of interest in fundamental pathophysiological studies and clinical trials. Current photoacoustic microscopy can noninvasively quantify properties of the microvasculature, including vessel density and diameter, with a high spatial resolution. However, the depth range of focus (i.e., focal zone) of optical-resolution photoacoustic microscopy (OR-PAM) is often insufficient to encompass the depth variations of features of interest—such as blood vessels—due to uneven tissue surfaces. Thus, time-consuming image acquisitions at multiple different focal planes are required to maintain the region of interest in the focal zone. We have developed continuous three-dimensional motorized contour-scanning OR-PAM, which enables real-time adjustment of the focal plane to track the vessels’ profile. We have experimentally demonstrated that contour scanning improves the signal-to-noise ratio of conventional OR-PAM by as much as 41% and shortens the image acquisition time by 3.2 times. Moreover, contour-scanning OR-PAM more accurately quantifies vessel density and diameter, and has been applied to studying tumors with uneven surfaces.

To research retinal stretching or distortion with accommodation, accommodation-induced changes in retinal thickness (RT) in the macular area were investigated in a population of young adults (n=23) by using a dual-channel spectral domain optical coherence tomography (SD-OCT) system manufactured in-house for this study. This dual-channel SD-OCT is capable of imaging the cornea and retina simultaneously with an imaging speed of 24 kHz A-line scan rate, which can provide the anatomical dimensions of the eye, including the RT and axial length. Thus, the modification of the RT with accommodation can be calculated. A significant decrease in the RT (13.50±1.25  μm) was observed during maximum accommodation. In the 4  mm×4  mm macular area centered at the fovea, we did not find a significant quadrant-dependent difference in retinal volume change, which indicates that neither retinal stretching nor distortion was quadrant-dependent during accommodation. We speculate that the changes in RT with maximum accommodation resulted from accommodation-induced ciliary muscle contractions.

In experimental stroke research, anesthesia is common and serves as a major reason for translational failure. Real-time cerebral blood flow (CBF) monitoring during stroke onset can provide important information for the prediction of brain injury; however, this is difficult to achieve in clinical practice due to various technical problems. We created a photothrombotic focal ischemic stroke model utilizing our self-developed miniature headstage in conscious and freely moving rats. In this model, a high spatiotemporal resolution imager using laser speckle contrast imaging technology was integrated to acquire real-time two-dimensional CBF information during thrombosis. The feasibility, stability, and reliability of the system were tested in terms of CBF, behavior, and T2-weighted magnetic resonance imaging (MRI) findings. After completion of occlusion, the CBF in the targeted cortex of the stroke group was reduced to 16±9% of the baseline value. The mean infarct volume measured by MRI 24 h postmodeling was 77±11  mm³ and correlated well with CBF (R²=0.74). This rodent model of focal cerebral ischemia and real-time blood flow imaging opens the possibility of performing various fundamental and translational studies on stroke without the influence of anesthetics.

Sensing and compensating of optical aberrations in closed-loop mode using a single spatial light modulator (SLM) for ophthalmic applications is demonstrated. Notwithstanding the disadvantages of the SLM, in certain cases, this multitasking capability of the device makes it advantageous over existing deformable mirrors (DMs), which are expensive and in general used for aberration compensation alone. A closed-loop adaptive optics (AO) system based on a single SLM was built. Beam resizing optics were used to utilize the large active area of the device and hence make it feasible to generate 137 active subapertures for wavefront sensing. While correcting Zernike aberrations up to fourth order introduced with the help of a DM (for testing purposes), diffraction-limited resolution was achieved. It is shown that matched filter and intensity-weighted centroiding techniques stand out among others. Closed-loop wavefront correction of aberrations in backscattered light from the eyes of three healthy human subjects was demonstrated after satisfactory results were obtained using an artificial eye, which was simulated with a short focal length lens and a sheet of white paper as diffuser. It is shown that the closed-loop AO system based on a single SLM is capable of diffraction-limited correction for ophthalmic applications.

Knowledge of light penetration characteristics is very important in almost all studies in biomedical optics. In this work, the reflectance sampling depth in biological tissues was investigated using Monte Carlo simulations for various common illumination/collection configurations. The analysis shows that the average sampling depth can be described by two simple empirical analytical expressions over the entire typical ranges of absorption and scattering properties relevant to in vivo biological tissue, regardless of the specific illumination/collection configuration details. These results are promising and helpful for the quick, efficient, and accurate design of reflectance studies for various biological tissue applications.

Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate the bladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degrades fluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studied their fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C). A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320/420  nm , FWHM=50/100  nm ) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455/525  nm , FWHM=80/50  nm ). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine’s main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370–430 nm to 395–415 nm would reduce the BWF background by a factor 2.

A temperature detection system using a micropipette thermocouple sensor was developed for use within mammalian cells during laser exposure with an 8.6-μm beam at 532 nm. We have demonstrated the capability of measuring temperatures at a single-cell level in the microscale range by inserting micropipette-based thermal sensors of size ranging from 2 to 4  μm into the membrane of a live retinal pigment epithelium (RPE) cell subjected to a laser beam. We setup the treatment groups of 532-nm laser-irradiated single RPE cell and in situ temperature recordings were made over time. Thermal profiles are given for representative cells experiencing damage resulting from exposures of 0.2 to 2 s. The measured maximum temperature rise for each cell ranges from 39 to 73°C; the RPE cells showed a signature of death for all the cases reported herein. In order to check the cell viability, real-time fluorescence microscopy was used to identify the transition of pigmented RPE cells between viable and damaged states due to laser exposure.

Despite the widespread use of radio frequency (RF) ablation, an effective way to assess thermal tissue damage during and after the procedure is still lacking. We present a method for monitoring RF ablation efficacy based on thermally induced methemoglobin as a marker for full tissue ablation. Diffuse reflectance (DR) spectra were measured from human blood samples during gradual heating of the samples from 37 to 60, 70, and 85°C. Additionally, reflectance spectra were recorded real-time during RF ablation of human liver tissue ex vivo and in vivo. Specific spectral characteristics of methemoglobin were extracted from the spectral slopes using a custom optical ablation ratio. Thermal coagulation of blood caused significant changes in the spectral slopes, which is thought to be caused by the formation of methemoglobin. The time course of these changes was clearly dependent on the heating temperature. RF ablation of liver tissue essentially led to similar spectral alterations. In vivo DR measurements confirmed that the method could be used to assess the degree of thermal damage during RF ablation and long after the tissue cooled.

The exact spatial distribution of impaired cerebral autoregulation in carotid artery disease is unknown. In this pilot study, we present a new approach of multichannel near-infrared spectroscopy (mcNIRS) for noninvasive spatial mapping of dynamic autoregulation in carotid artery disease. In 15 patients with unilateral severe carotid artery stenosis or occlusion, cortical hemodynamics in the bilateral frontal cortex were assessed from changes in oxyhemoglobin concentration using 52-channel NIRS (spatial resolution ∼2  cm). Dynamic autoregulation was graded by the phase shift between respiratory-induced 0.1 Hz oscillations of blood pressure and oxyhemoglobin. Ten of 15 patients showed regular phase values in the expected (patho) physiological range. Five patients had clearly outlying irregular phase values mostly due to artifacts. In patients with a regular phase pattern, a significant side-to-side difference of dynamic autoregulation was observed for the cortical border zone area between the middle and anterior cerebral artery ([i]p<0.05). In conclusion, dynamic cerebral autoregulation can be spatially assessed from slow hemodynamic oscillations with mcNIRS. In high-grade carotid artery disease, cortical dynamic autoregulation is affected mostly in the vascular border zone. Spatial mapping of dynamic autoregulation may serve as a powerful tool for identifying brain regions at specific risks for hemodynamic infarction.

A microcavity-based deoxyribonucleic acid (DNA) optical biosensor is demonstrated for the first time using synthetic sapphire for the optical cavity. Transmitted and elastic scattering intensity at 1510 nm are analyzed from a sapphire microsphere (radius 500  μm, refractive index 1.77) on an optical fiber half coupler. The 0.43 nm angular mode spacing of the resonances correlates well with the optical size of the sapphire sphere. Probe DNA consisting of a 36-mer fragment was covalently immobilized on a sapphire microsphere and hybridized with a 29-mer target DNA. Whispering gallery modes (WGMs) were monitored before the sapphire was functionalized with DNA and after it was functionalized with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The shift in WGMs from the surface modification with DNA was measured and correlated well with the estimated thickness of the add-on DNA layer. It is shown that ssDNA is more uniformly oriented on the sapphire surface than dsDNA. In addition, it is shown that functionalization of the sapphire spherical surface with DNA does not affect the quality factor (Q≈10⁴) of the sapphire microspheres. The use of sapphire is especially interesting because this material is chemically resilient, biocompatible, and widely used for medical implants.

Microfracture surgery is a bone marrow stimulation technique for treating cartilage defects and injuries in the knee. Current methods rely on surgical skill and instrumentation. This study investigates the potential use of laser technology as an alternate means to create the microfracture holes. Lasers investigated in this study include an erbium:YAG laser (λ=2.94  μm ), titanium:sapphire femtosecond laser system (λ=1700  nm ), and Nd:glass femtosecond laser (λ=1053  nm ). Bovine samples were ablated at fluences of 8 to 18  J/cm² with the erbium:YAG laser, at a power of 300±15  mW with the titanium:sapphire femtosecond system, and at an energy of 3  μJ/pulse with the Nd:glass laser. Samples were digitally photographed and histological sections were taken for analysis. The erbium:YAG laser is capable of fast and efficient ablation; specimen treated with fluences of 12 and 18  J/cm² experienced significant amounts of bone removal and minimal carbonization with saline hydration. The femtosecond laser systems successfully removed cartilage but not clinically significant amounts of bone. Precise tissue removal was possible but not to substantial depths due to limitations of the systems. With additional studies and development, the use of femtosecond laser systems to ablate bone may be achieved at clinically valuable ablation rates.

We evaluated the effect of different irradiation parameters in low-level laser therapy (LLLT) for treating inflammation induced in the gastrocnemius muscle of rats through cytokines concentration in systemic blood and analysis of muscle tissue. We used continuous (830 and 980 nm) and pulsed illuminations (830 nm). Animals were divided into five groups per wavelength (10, 20, 30, 40, and 50 mW), and a control group. LLLT was applied during 5 days with a constant irradiation time and area. TNF-α , IL-1β , IL-2, and IL-6 cytokines were quantified by ELISA. Inflammatory cells were counted using microscopy. Identical methodology was used with pulsed illumination. Average power (40 mW) and duty cycle were kept constant (80%) at five frequencies (5, 25, 50, 100, and 200 Hz). For continuous irradiation, treatment effects occurred for all doses, with a reduction of TNF-α , IL-1β , and IL-6 cytokines and inflammatory cells. Continuous irradiation at 830 nm was more effective, a result explained by the action spectrum of cytochrome c oxidase (CCO). Best results were obtained for 40 mW, with data suggesting a biphasic dose response. Pulsed wave irradiation was only effective for higher frequencies, a result that might be related to the rate constants of the CCO internal electron transfer process.

Light dosimetry is an important parameter that affects the efficacy of photodynamic therapy (PDT). However, the irregular morphologies of lesions complicate lesion segmentation and light irradiance adjustment. Therefore, this study developed an illumination demo system comprising a camera, a digital projector, and a computing unit to solve these problems. A three-dimensional model of a lesion was reconstructed using the developed system. Hierarchical segmentation was achieved with the superpixel algorithm. The expected light dosimetry on the targeted lesion was achieved with the proposed illumination procedure. Accurate control and optimization of light delivery can improve the efficacy of PDT.

Photoimmunotherapy (PIT) is a cell-specific cancer therapy based on an armed antibody conjugate that induces rapid and highly selective cancer cell necrosis after exposure to near-infrared (NIR) light. The PIT treatment also induces the superenhanced permeability and retention effect, which allows high concentrations of nanoparticles to accumulate in the tumor bed. In our pilot studies, optical coherence tomography (OCT) reveals dramatic hemodynamic changes during PIT. We developed and applied speckle variance analysis, Doppler flow measurement, bulk motion removal, and automatic region of interest selection to quantify vessel diameter and blood velocity within tumors in vivo. OCT imaging reveals that blood velocity in peripheral tumor vessels quickly drops below the detection limit while the vessel lumen remains open (4 vessels from 3 animals). On the other hand, control tumor vessels (receive NIR illumination but no PIT drug) do not show the sustained blood velocity drop (5 vessels from 3 animals). Ultraslow blood velocity could result in a long drug circulation time in tumor. Increase of the blood pool volume within the central tumor (shown in histology) may be the leading cause of the periphery blood velocity drop and could also increase the drug pool volume in tumor vessels.